Methods of making surfactant and cleaning compositions through microbially produced branched fatty alcohols

ABSTRACT

The invention provides a surfactant and/or a cleaning composition comprising a microbially produced branched fatty alcohol or a derivative thereof. The invention also provides a household cleaning composition and a personal or pet care cleaning composition comprising a microbially produced branched fatty alcohol or a derivative thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Pat. No. 8,859,259, filedFeb. 14, 2011, which claims the benefit of U.S. Provisional PatentApplication No. 61/304,448, filed Feb. 14, 2010, and U.S. ProvisionalPatent Application No. 61/324,310, filed Apr. 15, 2010, the entirecontents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Fatty alcohols have many commercial uses. Worldwide annual sales offatty alcohols and their derivatives are in excess of US$1 billion.Fatty alcohols are used in diverse industries. For example, they areused in the cosmetic and food industries as emulsifiers, emollients, andthickeners. Due to their amphiphilic nature, fatty alcohols can beformulated or be used per se as nonionic surfactants, which are usefulin personal care and household products, for example, in detergents. Inaddition, fatty alcohols are used in waxes, gums, resins, pharmaceuticalsalves and lotions, lubricating oil additives, textile antistatic andfinishing agents, plasticizers, cosmetics, industrial solvents, andsolvents for fats.

One major use for fatty alcohols is in cleaning compositions. On theother hand, fatty alcohols find applicability as surfactants, which are,for example, capable of enhancing oil recovery and/or engineperformance. Conventional surfactants comprise molecules having at leastone water-solubilizing substituent or moiety (e.g., hydrophilic group)and at least one oleophilic substituent or moiety (e.g., hydrophobicgroup). Examples of hydrophilic groups include, without limitation,carboxylate, sulfate, sulfonate, amine oxide, or polyoxyethylene.Examples of the hydrophobic groups include, without limitation, alkyl,alkenyl, or alkaryl hydrophobes, which typically contain about 10 toabout 20 carbon atoms.

Surfactants are typically regarded as the major force behind cleaningproducts' ability to break up stains, solubilize dirt and soil, and/orprevent their redeposition to surfaces. As such, surfactants are alsoreferred to as wetting agents and foamers, which lower the surfacetension of the medium in which they are dissolved. Capable of loweringthe interfacial tension between two media or interfaces (e.g.,air/water, water/oil, or oil/solid interfaces), surfactants play a keyrole, and are often the most important component in detergents.Conventional detergent compositions contain mixtures of varioussurfactants in order to remove different types of soils and stains fromsurfaces.

The earliest utilized source of hydrophobe groups were natural fats andoils, which were converted into soaps (e.g., carboxylate hydrophile)using base via saponification processes. Coconut and palm oils are tothis day used to manufacture soaps and alkylsulfate surfactants. Asedible oils became more scarce, it has become increasingly prevalent tomanufacture detergents from petrochemicals, using processes such as theZeigler process to convert petroleum derived ethylene to fatty alcohols.For example, ethylene has been converted into alkyl benezene sulfonatesurfactants, which are commonly found in today's detergents and cleaningcompositions.

Fatty alcohols can also served as starting materials in the preparationof surfactants and of other cleaning composition ingredients including,for example, alkyl sulfates, fatty ether sulfates, fatty alcoholsulfates, fatty phosphate esters, alkylbenzyl dimethylammonium salts,fatty amine oxides, alkyl polyglucosides, and alkyl glyceryl ethersulfonates. Among these, alkyl sulfates are commonly known due to theease of their manufacture as well as their improved solubility andsurfactant characteristics over traditional soap-based surfactants.However, long-chain alkyl surfactants have less than optimal performanceas surfactants or as component(s) of detergents at low temperatures(e.g., about 50° C. or lower, about 30° C. or lower).

While there have been isolated reports that branching, especiallytowards the middle part of the long-chain alkyl, can reduce solubilityof the surfactant, others have described that, in commercial practices,branching in fatty alcohols is highly desirable. See, e.g., R. G.Laughlin, The Aqueous Phase Behavior of Surfactants,” Academic Press,N.Y., (1994), at page 347; but see, Finger et al., Detergentalcohols—the effect of alcohol structure and molecular weight onsurfactant properties, J. Amer. Oil Chemicals Society, Vol. 44:525(1967); Technical Bulletin, Shell Chemical Co., SC:164-80. In addition,K. R. Wormuth, et al., Langmuir, vol 7 (1991):2048-2053, describes thetechnical advantages observed with a number of branched alkyl sulfates,especially with the “branched Guerbet” type, derived from the highlybranched “Exxal” alcohols (Exxon). Phase studies have established aliphophilic ranking (i.e., a hydrophobicity ranking) if highlybranched/double tail>methyl branched>linear. Furthermore, patents andapplications, including, for example, U.S. Pat. No. 6,008,181 indicatesthat certain branched or multi-branched fatty alcohol derivativesexhibit improved cleaning capacity, especially at lower temperatures.

Branched fatty alcohols and various precursors are known to haveadditional preferred properties such as considerably lower meltingpoints, which can in turn confer lower pour points when made intoindustrial chemicals, as compared to linear alcohols of comparablemolecular weights. They are also known to confer substantially lowervolatility and vapor pressure, and improved stability against oxidationand rancidity than their linear counterparts. These additional preferredproperties, in addition to making branched materials desirablesurfactants, make them particularly suited as components or feedstocksfor cosmetic and pharmaceutical applications, as components ofplasticizers for making synthetic resins, as solvents for solutions forprinting ink and specialty inks, or as industrial lubricants.

Those added preferred properties can be alternatively obtained fromunsaturated fatty alcohols and precursors. But unsaturation promotesoxidation, leading to short shelf lives and corrosion. Thus desirableproperties, e.g., lower melting points, pour points, volatility, andvapor pressure and improved oxidative stability, are better achieved viabranching.

Obtaining branched materials from crude petroleum requires a significantfinancial investment as well as consumes a great deal of energy. It isalso an inefficient process because frequently it is necessary to crackthe long chain hydrocarbons in crude petroleum to produce smallermonomers, which only then become useful as raw materials formanufacturing complex specialty chemicals. Furthermore, it iscommonplace in the petrochemical industry to obtain branched chemicals,such as branched alcohols and aldehydes, by isomerization ofstraight-chain hydrocarbons. Expensive catalysts are typically requiredfor isomerization, thus increasing manufacturing cost. The catalystsoften then become undesirable contaminants that are removed from thefinished products, adding yet further cost to the processes.

Obtaining specialty chemicals such as branched alcohols or derivativesfrom crude petroleum also drains the dwindling resource of petroleum, inaddition to the cost and problems associated with exploring, extracting,transporting, and refining. One estimate of world petroleum consumptionis 30 billion barrels per year. By some estimates, it is predicted thatat current production levels, the world's petroleum reserves could bedepleted before 2050.

Finally, processing and manufacturing of surfactants and/or detergentsfrom petroleum inevitably releases greenhouse gases (e.g., in the formof carbon dioxide) and other forms of air pollution (e.g., carbonmonoxide, sulfur dioxide, etc.). The accumulation of greenhouse gases inthe atmosphere can lead to increase global warming, causing localpollutions and spillage as well as global environmental detriments.

Thus, although it is possible to obtain branched fatty alcohols andderivatives from natural oils and petroleum, it would be desirable toproduce these branched materials from other sources, such as directlyfrom biomass.

SUMMARY OF THE INVENTION

The invention provides a surfactant composition and a cleaningcomposition comprising one or more microbially produced branched fattyalcohols, branched fatty alcohol precursors, or branched fatty alcoholderivatives thereof.

The invention provides a surfactant composition comprising about 0.001wt. % to about 100 wt. % of one or more microbially produced branchedfatty alcohols or branched alcohol derivatives thereof.

The invention also provides a liquid cleaning composition comprising (a)about 0.1 wt. % to about 50 wt. % of one or more microbially producedbranched fatty alcohols or derivatives thereof, or about 0.1 wt. % toabout 50 wt. % of a surfactant comprising one or more microbiallyproduced branched fatty alcohols or derivatives thereof, (b) about 1 wt.% to about 30 wt. % of one or more co-surfactants, (c) about 0 wt. % toabout 10 wt. % of one or more detergency builders, (d) 0 wt. % to about2 wt. % of one or more enzymes, (e) about 0 wt. % to about 15 wt. % ofone or more chelating agents, (f) about 0 wt. % to about 20 wt. % of oneor more hydrotropes, (g), about 0 wt. % to about 1.0 wt. % of one ormore organic sequestering agents, and (h) about 0.1 wt. % to about 98wt. % of a solvent system. In some embodiments, the liquid cleaningcomposition further comprises one or more suitable adjuncts.

The invention further provides a solid cleaning composition comprising(a) about 0.1 wt. % to about 50 wt. % of one or more microbiallyproduced branched fatty alcohols or derivatives thereof, or about 0.1wt. % to about 50 wt. % of a surfactant comprising one or moremicrobially produced branched fatty alcohols or derivatives thereof, (b)about 1 wt. % to about 30 wt. % of one or more co-surfactants, (c) about1 wt. % to about 60 wt. % of one or more detergency builders, (d) about0 wt. % to about 2 wt. % of one or more enzymes, (e) about 0 wt. % toabout 20 wt. % of one or more hydrotropes, (f) about 10 wt. % to about35 wt. % of one or more filler salts, (g) about 0 wt. % to about 15 wt.% of one or more chelating agents, and (g) about 0.01 wt. % to about 1wt. % of one or more organic sequestering agents. In certainembodiments, the solid cleaning composition further comprises one ormore suitable adjuncts.

In particular embodiments, the invention pertains to a householdcleaning composition comprising (a) about 0.1 wt. % to about 50 wt. % ofone or more microbially produced branched fatty alcohols and/orderivatives thereof, or about 0.1 wt. % to about 50 wt. % of asurfactant comprising one or more microbially produced fatty alcoholsand/or derivatives thereof; (b) about 1 wt. % to about 30 wt. % of oneor more co-surfactants; (c) about 0 wt. % to about 30 wt. % of one ormore detergency builders; (d) about 0 wt. % to about 2.0 wt. % of one ormore suitable detersive enzymes; (e) about 0 wt. % to about 15 wt. % oneor more chelating agents; (f) about 0 wt. % to about 20 wt. % of one ormore hydrotropes, (g) about 0 to about 15 wt. % of one or more rheologymodifier; (h) about 0 wt. % to about 1.0 wt. % of one or more organicsequestering agents; and (i) various other adjuncts such as, forexample, one or more of bleaching agents, additional enzymes, sudssuppressors, dispersants, lime-soap dispersants, soil suspension andanti-redeposition agents, and corrosion inhibitors. In an exemplaryembodiment, a laundry composition can also comprise softening agents,fragrances, bleach systems, dyes or colorants, preservatives,germicides, fungicides, fabric care benefit agents, gelling agents,antideposition agents, and other detersive adjuncts

Such a household cleaning composition can be a liquid, which furthercomprises water and/or a suitable aqueous carrier or solvent. Liquidcompositions can be in a “concentrated” form, the density of which canrange from, for example, about 400 to about 1,200 g/L, when measured at200° C. For example, the water content of a typical concentrated liquiddetergent is less than about 40 wt. %, or less than about 30 wt. %.Alternatively, a household cleaning composition can be a solid, forexample, in the form of a tablet, a bar, a powder or a granule. Granularcompositions can also be in a “compact” form, which is best reflected bydensity and, in terms of composition, by the amount of inorganic fillersalt. Inorganic filler salts are conventional ingredients of solidcleaning compositions, present in substantial amounts, varying from, forexample, about 10 wt. % to about 35 wt. %. Suitable filler saltsinclude, for example, alkali and alkaline-earth metal salts of sulfatesand chlorides. An exemplary filler salt is sodium sulfate.

In another embodiment, the invention provides a personal or beauty carecleaning or treatment composition comprising (a) about 0.1 wt. % toabout 50 wt. % of one or more microbially produced branched fattyalcohols and/or derivatives thereof, or about 0.1 wt. % to about 50 wt.% of a surfactant comprising one or more microbially produced branchedfatty alcohols and/or derivatives thereof; (b) about 0.001 wt. % toabout 30 wt. % of one or more co-surfactants; (c) about 0 wt. % to about30 wt. % of one or more detergency builders; (d) about 0 wt. % to about2.0 wt. % of one or more suitable detersive enzymes; (e) about 0 wt. %to about 15 wt. % one or more chelating agents; (f) about 0 wt. % toabout 20 wt. % of one or more hydrotropes, (g) about 0 to about 15 wt. %of one or more rheology modifier; (h) about 0 wt. % to about 1.0 wt. %of one or more organic sequestering agents; and (i) various otheradjuncts such as, for example, one or more of conditioner, silicone,fragrances, silica particles, cationic cellose or guar polymers,silicone microemulsion stabilizers, fatty amphiphiles, germicides,fungicides, anti-dandruff agents, pearlescent agents, foam boosters,pediculocides, pH adjusting agents, UV absorbers, sunscreens, skinactive agents, vitamins, minerals, herbal/fruit/food extracts,sphingolipids, sensory indicators, suspension agents, and mixturesthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B are schematics of two exemplary alternative pathwaysfor producing branched fatty alcohols using recombinant microbial hostcells.

FIG. 2A lists representative homologs of BKD E1 alpha subunit, theiramino acid sequences and polynucleotide sequences, as well as amino acidsequence motifs of suitable BKD E1 alpha subunit homologs and variants.FIG. 2B lists representative homologs of BKD E1 beta subunit, theiramino acid sequences and polynucleotide sequences, as well as amino acidsequence motifs of suitable BKD E1 beta subunit homologs and variants.FIG. 2C lists representative homologs of BKD E2 subunit, their aminoacid sequences and polypeptide sequences, as well as amino acid sequencemotifs of suitable BKD E2 subunit homologs and variants. FIG. 2D listsrepresentative homologs of BKD E3 subunit homologs and variants, as wellas amino acid sequence motifs of suitable BKD E3 subunit homologs andvariants. FIG. 2E lists representative homologs of beta ketoacyl-ACPsynthase homologs, their amino acid sequences and polynucleotidesequences, as well as amino acid sequences of suitable betaketo-acyl-ACP synthase homologs and variants.

FIG. 3A is a table of BKD E1 alpha subunit homologs. FIG. 3B is a tableof BKD E1 beta subunit homologs. FIG. 3C is a table of BKD E2 subunithomologs. FIG. 3D if a table of BKD E3 subunit homologs. FIG. 3E is atable of beta ketoacyl-ACP synthase homologs. These tables also present% identity in reference to the sequences of various organisms. Forexample, “ID % Pp” indicates that the identity listed in the columnbelow are in reference to a P. putida gene encoding that subunit. “ID %Bs” refers to the identity to a B. subtilis gene encoding that subunit.“ID % Sc” and “ID % Sc2” refer to identity to a first and second S.coelicolor genes encoding that subunit, respectively. “ID % Sa” and “ID% Sa2” refer to identity to a first and a second S. avermitilis genesencoding that subunit, respectively.

FIG. 4A depicts a GC/MS trace of branched fatty alcohol production ofstrain MG1655_(—) ΔtonA AAR:kan transformed with a pGL10 vectorcontaining P. putida Pput1450, Pput1451, Pput1452 and Pput1453 inserts,and with B. subtilis fabH1. The figure indicates the production ofiso-C_(14:0), iso-C_(15:0), anteiso-C_(15:0), iso-C_(16:0), iso-C_(17:0)and anteiso-C_(17:0) branched fatty alcohols. FIG. 4B depicts theproduction of branched fatty acyl-CoA precursors by feeding branchedsubstrates isobutyrate and isovalerate to an engineered E. coli straincomprising the pDG10 and an OP-180 plasmids, the latter plasmidcontained teas under the control of a Ptrc promoter.

FIG. 5 is a representative calibration curve obtained by linearregression, which was used in the semi-quantitative measurement of theamount of branched fatty alcohol yield relative to the amount ofstraight-chain fatty alcohol yield.

FIG. 6A is a listing of nucleotide sequence of the pDG2 plasmid. FIG. 6Bdepicts a map of the pDG6 plasmid. FIG. 6C is a listing of nucleotidesequence of the pDG6 plasmid, constructed by inserting B. subtilis fabH1into pDG2, comprising E. coli PfabH1 (promoter) and B. subtilis fabH1.The B. subtilis fabH1 insert is in upper case italic letters. FIG. 6Ddepicts a map of the pDG7 plasmid. FIG. 6E is a listing of nucleotidesequence of the pDG7 plasmid, constructed by inserting a B. subtilisfabH2 into pDG2, comprising E. coli PfabH1 (promoter) and B. subtilisfabH2. FIG. 6F depicts a map of the pDG8 plasmid. FIG. 6G is a listingof nucleotide sequence of pDG8 plasmid, constructed by inserting S.coelicolor fabH into pDG2, comprising E. coli PfabH1 (promoter) and S.coelicolor fabH. FIG. 6H is a plasmid map of the pDG10 plasmid. FIG. 6Iis listing of nucleotide sequence of the pDG10 plasmid, comprising a C.acetobutylicum ptb_buk insert. FIG. 6J is a listing of nucleotidesequence of the pLS9-111 plasmid. FIG. 6K is a listing of nucleotidesequence of the pLS9-114 plasmid. FIG. 6L is a listing of nucleotidesequence of the pLS9-115 plasmid.

FIG. 7 is a listing of nucleotide sequence of the pKZ4 plasmid having apGL10.173B vector backbone and a polynucleotide insert encoding a BKDcomplex from Pseudomonas putida. The P. putida genes encoding a BKDcomplex are shown in lower case italic letters.

FIG. 8 is a listing of nucleotide sequence of the pGL10.173B vectorbackbone, which contains the BamHI and EcoRI sites to which thePseudomonas putida bkd genes (operon) were inserted. The BamHI and EcoRIrestriction sites are marked.

FIG. 9 is a listing of additional nucleotide and amino acid sequences ofthe disclosure.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein, including GenBank database sequences,are incorporated by reference in their entirety. In case of conflict,the present specification, including definitions, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DEFINITIONS

Throughout the specification, a reference may be made using anabbreviated gene name or polypeptide name, but it is understood thatsuch an abbreviated gene or polypeptide name represents the genus ofgenes or polypeptides. Such gene names include all genes encoding thesame polypeptide and homologous polypeptides having the samephysiological function. Polypeptide names include all polypeptides andhomologous polypeptides that have the same activity (e.g., that catalyzethe same fundamental chemical reaction).

Unless otherwise indicated, the accession numbers referenced herein arederived from the NCBI database (National Center for BiotechnologyInformation) maintained by the National Institute of Health, U.S.A.Unless otherwise indicated, the accession numbers are as provided in thedatabase as of December 2009.

EC numbers are established by the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology (NC-IUBMB)(available at http://www.chem. qmul.ac.uk/iubmb/enzyme/). The EC numbersreferenced herein are derived from the KEGG Ligand database, maintainedby the Kyoto Encyclopedia of Genes and Genomics, sponsored in part bythe University of Tokyo. Unless otherwise indicated, the EC numbers areas provided in the database as of October 2008.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” is used herein to mean a value ±20% of a givennumerical value. Thus, “about 60%” means a value of between 60±(20% of60) (i.e., between 48 and 70).

The term “alkyl” is used herein to mean a straight chain or a branchedchain hydrocarbon residue having from about 6 carbon atoms to about 26carbon atoms and in the context of the present specification is usedinterchangeably with the term “fatty.”

As used herein, the term “alcohol dehydrogenase” (EC 1.1.1.*) refers toa polypeptide capable of catalyzing the conversion of a fatty aldehydeto an alcohol (e.g., fatty alcohol). In certain embodiments, theseenzymes can also be referred to as fatty aldehyde recutases,oxidoreductases, or aldo-keto reductases. Additionally, one of ordinaryskill in the art will appreciate that some alcohol dehydrogenases willcatalyze other reactions as well. For example, some alcoholdehydrogenases will accept other substrates in addition to fattyaldehydes. Such non-specific alcohol dehydrogenases are, therefore, alsoincluded in this definition. Nucleic acid sequences encoding alcoholdehydrogenases are known in the art, and such alcohol dehydrogenases arepublicly available. Exemplary GenBank Accession Numbers are provided inTable 8 herein.

As used herein, the term “attenuate” means to weaken, reduce, ordiminish. For example, a polypeptide can be attenuated by modifying thepolypeptide to reduce its activity (e.g., by modifying a nucleotidesequence that encodes the polypeptide) or its expression level.

As used herein, the term “biomass” refers to any biological materialfrom which a carbon source is derived. In some instances, a biomass isprocessed into a carbon source, which is suitable for bioconversion. Inother instances, the biomass may not require further processing into acarbon source. The carbon source can be converted into a fatty alcohol.One exemplary source of biomass is plant matter or vegetation. Forexample, corn, sugar cane, or switchgrass can be used as biomass.Another non-limiting example of biomass is metabolic wastes, such asanimal matter, for example cow manure. In addition, biomass may includealgae and other marine plants. Biomass also includes waste products fromindustry, agriculture, forestry, and households. Examples of such wasteproducts that can be used as biomass are fermentation waste, ensilage,straw, lumber, sewage, garbage, cellulosic urban waste, and foodleftovers. Biomass also includes carbon sources such as carbohydrates(e.g., monosaccharides, disaccharides, or polysaccharides).

As used herein, the phrase “carbon source” refers to a substrate orcompound suitable to be used as a source of carbon for prokaryotic orsimple eukaryotic cell growth. Carbon sources can be in various forms,including, but not limited to polymers, carbohydrates, acids, alcohols,aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO₂).These include, for example, various monosaccharides, such as glucose,fructose, mannose, and galactose; oligosaccharides, such asfructo-oligosaccharide and galacto-oligosaccharide; polysaccharides suchas xylose and arabinose; disaccharides, such as sucrose, maltose, andturanose; cellulosic material, such as methyl cellulose and sodiumcarboxymethyl cellulose; saturated or unsaturated fatty acid esters,such as succinate, lactate, and acetate; alcohols, such as ethanol,methanol, and glycerol, or mixtures thereof. The carbon source can alsobe a product of photosynthesis, including, but not limited to, glucose.A preferred carbon source is biomass. Another preferred carbon source isglucose.

A nucleotide sequence is “complementary” to another nucleotide sequenceif each of the bases of the two sequences matches (i.e., is capable offorming Watson-Crick base pairs). The term “complementary strand” isused herein interchangeably with the term “complement”. The complementof a nucleic acid strand can be the complement of a coding strand or thecomplement of a non-coding strand.

As used herein, the term “conditions sufficient to allow expression”means any conditions that allow a host cell to produce a desiredproduct, such as a polypeptide or fatty alcohol described herein.Suitable conditions include, for example, fermentation conditions.Fermentation conditions can comprise many parameters, such astemperature ranges, levels of aeration, and media composition. Each ofthese conditions, individually and in combination, allows the host cellto grow. Exemplary culture media include broths or gels. Generally, themedium includes a carbon source, such as glucose, fructose, cellulose,or the like, that can be metabolized by a host cell directly. Inaddition, enzymes can be used in the medium to facilitate themobilization (e.g., the depolymerization of starch or cellulose tofermentable sugars) and subsequent metabolism of the carbon source.

To determine if conditions are sufficient to allow expression, a hostcell can be cultured, for example, for about 4, 8, 12, 24, 36, or 48hours. During and/or after culturing, samples can be obtained andanalyzed to determine if the conditions allow expression. For example,the host cells in the sample or the medium in which the host cells weregrown can be tested for the presence of a desired product. When testingfor the presence of a product, assays, such as TLC, HPLC, GC/FID, GC/MS,LC/MS, and MS, can be used.

It is understood that the polypeptides described herein may haveadditional conservative or non-essential amino acid substitutions, whichdo not have a substantial effect on the polypeptide functions. Whetheror not a particular substitution will be tolerated (i.e., will notadversely affect desired biological properties, such as carboxylic acidreductase activity) can be determined as described in Bowie et al.,Science, 247: 1306-1310 (1990). A “conservative amino acid substitution”refers to the replacement of one amino acid residue with another aminoacid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine), and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

As used herein, “control element” means a transcriptional and/or atranslational control element. Control elements include promoters andenhancers, such as ribosome binding sequences. The term “promoterelement,” “promoter,” or “promoter sequence” refers to a DNA sequencethat functions as a switch that activates the expression of a gene. Ifthe gene is activated, it is said to be transcribed or participating intranscription. Transcription involves the synthesis of mRNA from thegene. A promoter, therefore, serves as a transcriptional regulatoryelement and also provides a site for initiation of transcription of thegene into mRNA. Control elements interact specifically with cellularproteins involved in transcription (Maniatis et al., Science, 236: 1237(1987)).

As used herein, the term “detergent” refers broadly to agents andmaterials that are useful in cleaning applications or as cleaning aids.This term is thus used interchangeably with the term “cleaningcomposition.” The term encompasses materials and agents that comprisevarious surfactants at various percentages by weight or by volume, aswell as suitable additives, and are capable of emulsifying stains in acleaning matrix. A detergent can take the physical form of, for example,a liquid, a paste, a gel, a bar, a powder, a tablet, or a granule.Granular compositions can also be in “compact” form, whereas liquidcompositions can be in “concentrate” form.

As used herein, detergent compositions include articles and compositionsof cleaning and/or treatment. As used herein, the term “cleaning and/ortreatment composition” includes, unless otherwise indicated, tablet,granular, or power-form all-purpose or “heavy duty” washing agents,especially laundry detergents; liquid, gel, or paste-form all-purposewashing agents, especially the so-called heavy-duty liquid types; liquidfine-fabric detergents; hand dishwashing agents, or light dutydishwashing agents, especially those of the high-foaming type; machinedishwashing agents, including the various tablets, granular, liquid andrinse-aid types for household and institutional use. The compositionscan also be in unit dose packages, including those known in the art andthose that are water soluble, water insoluble and/or water permeable.

As used herein, detergent composition also include personal or beautycare products in the form of skin and hair care compositions including,for example, conditioning treatments, cleansing products, such as hairand/or scalp shampoos, body washes, hand cleaners, water-less handsanitizers/cleansers, facial cleansers, and the like.

As used herein, the term “fatty acid” means a carboxylic acid having theformula RCOOH. R represents an aliphatic group, preferably an alkylgroup. R can comprise about 4 or more carbon atoms. In some embodiments,the fatty acid comprises between about 4 and about 22 carbon atoms.Fatty acids can be saturated, monounsaturated, or polyunsaturated. Inaddition, fatty acids can comprise a straight or branched chain. Thebranched chains may have one or more points of branching. In addition,the branched chains may include cyclic branches. In a preferredembodiment, the fatty acid is made from a fatty acid biosyntheticpathway.

As used herein, the term “fatty acid biosynthetic pathway” means abiosynthetic pathway that produces fatty acids. The fatty acidbiosynthetic pathway includes fatty acid enzymes that can be engineered,as described herein, to produce fatty acids, and in some embodiments canbe expressed with additional enzymes to produce fatty acids havingdesired carbon chain characteristics.

As used herein, the term “fatty acid derivative” means products made inpart from the fatty acid biosynthetic pathway of the production hostorganism. “Fatty acid derivative” also includes products made in partfrom acyl-ACP or acyl-ACP derivatives. The fatty acid biosyntheticpathway includes fatty acid synthase enzymes which can be engineered asdescribed herein to produce fatty acid derivatives, and in some examplescan be expressed with additional enzymes to produce fatty acidderivatives having desired carbon chain characteristics. Exemplary fattyacid derivatives include, for example, fatty acids, acyl-CoA, fattyaldehyde, short and long chain alcohols, hydrocarbons, fatty alcohols,and esters (e.g., waxes, fatty acid esters, or fatty esters), althoughdue to their separate and industrial utilities and depending the sourcesfrom which they derive, hydrocarbons can sometimes be grouped into aseparate “hydrocarbon” category.

As used herein, the term “fatty acid derivative enzyme” means any enzymethat may be expressed or overexpressed in the production of fatty acidderivatives. These enzymes may be part of the fatty acid biosyntheticpathway. Non-limiting examples of fatty acid derivative enzymes includefatty acid synthases, thioesterases, acyl-CoA synthases, acyl-CoAreductases, alcohol dehydrogenases, alcohol acyltransferases, fattyalcohol-forming acyl-CoA reductases, carboxylic acid reductases (e.g.,fatty acid reductases), acyl-ACP reductases, fatty acid hydroxylases,acyl-CoA desaturases, acyl-ACP desaturases, acyl-CoA oxidases, acyl-CoAdehydrogenases, ester synthases, and/or alkane biosyntheticpolypeptides, etc. Fatty acid derivative enzymes can convert a substrateinto a fatty acid derivative. In some examples, the substrate may be afatty acid derivative that the fatty acid derivative enzyme convertsinto a different fatty acid derivative.

As used herein, “fatty acid enzyme” means any enzyme involved in fattyacid biosynthesis. Fatty acid enzymes can be expressed or overexpressedin host cells to produce fatty acids. Non-limiting examples of fattyacid enzymes include fatty acid synthases and thioesterases.

As used herein, “fatty aldehyde” means an aldehyde having the formulaRCHO characterized by an unsaturated carbonyl group (C═O). In apreferred embodiment, the fatty aldehyde is any aldehyde made from afatty acid or fatty acid derivative. In one embodiment, the R group isat least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbons in length, or is avalue between any two of the foregoing values.

R can be straight or branched chain. The branched chains may have one ormore points of branching. In addition, the branched chains may includecyclic branches.

Furthermore, R can be saturated or unsaturated. If unsaturated, the Rcan have one or more points of unsaturation.

In one embodiment, the fatty aldehyde is produced biosynthetically.

Fatty aldehydes have many uses. For example, fatty aldehydes can be usedto produce many specialty chemicals. For example, fatty aldehydes areused to produce polymers, resins, dyes, flavorings, plasticizers,perfumes, pharmaceuticals, and other chemicals. Some are used assolvents, preservatives, or disinfectants. Some natural and syntheticcompounds, such as vitamins and hormones, are aldehydes.

The terms “fatty aldehyde biosynthetic polypeptide”, “carboxylic acidreductase”, and “CAR” are used interchangeably herein.

As used herein, “fatty alcohol” means an alcohol having the formula ROH.In a preferred embodiment, the fatty alcohol is any alcohol made from afatty acid or fatty acid derivative. In one embodiment, the R group isat least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, or 26 carbons in length, or is a value betweenany two of the foregoing values. Typically, the fatty alcohol comprisesan R group that is 6 to 26 carbons in length. Preferably, the fattyalcohol comprises an R group that is 8, 10, 12, 14, 16, or 18 carbons inlength.

R can be straight or branched chain. The branched chains may have one ormore points of branching. In addition, the branched chains may includecyclic branches. In a particular embodiment, the fatty alcohol of thepresent invention comprises one or more points of branching.

Furthermore, R can be saturated or unsaturated. If unsaturated, the Rcan have one or more points of unsaturation.

In one embodiment, the branched fatty alcohol is producedbiosynthetically.

Fatty alcohols have many uses. For example, fatty alcohols can be usedto produce various specialty chemicals. As such, fatty alcohols are usedas a biofuel; as solvents for fats, waxes, gums, and resins; inpharmaceutical salves, emollients, and lotions; as lubricating-oiladditives; in detergents and emulsifiers; as textile antistatic andfinishing agents; as plasticizers; as nonionic surfactants; incosmetics, e.g., as thickeners.

The term “fatty alcohol derivative” refers to a compound derived from afatty alcohol. The fatty alcohol derivative can include the oxygen atomderived from the fatty alcohol, or, in some embodiments, does notinclude the aforesaid oxygen atom, in, for example, fatty amine oxides.For example, a fatty amide, which also can be referred to as an alkylamide, refers to a compound comprising an amide group and a hydrocarbonresidue having about 6 carbon atoms or more, wherein the hydrocarbonresidue is bonded to the carbonyl group of the amide group or to thenitrogen atom of the amide group. In some embodiments, the hydrocarbonresidue of the fatty alcohol is bonded to the carbonyl group of theamide group or to the nitrogen atom of the amide group. In someembodiments, the hydrocarbon residue is saturated. In other embodiments,the hydrocarbon residue is monounsaturated. In further embodiments, thehydrocarbon residue is polyunsaturated. In certain other embodiments,the hydrocarbon residue can be a straight-chain residue. In certainfurther embodiments, the hydrocarbon residue can contain one or morepoints of branching.

Branched fatty alcohols have particularly beneficial properties ascompared to their corresponding straight-chain isomers (i.e., isomers ofthe same molecular weight). For example, branched fatty alcohols tend tohave considerably lower melting points when compared to theircorresponding straight-chain isomers. Lower melting points confer lowerpour points. In addition, branched fatty alcohols tend to substantiallylower volatility and vapor pressure, and improved stability againstoxidation and rancidity, as compared to their correspondingstraight-chain isomers. These beneficial properties render particularsuitability of using branched fatty alcohols and/or derivatives thereofas components or feedstocks for cosmetic and pharmaceuticalapplications, as components of plasticizers for synthetic resins, assolvents for solutions for printing ink and specialty inks, or asindustrial lubricants. These materials are also well suited ascomponents of surfactants that have good low-temperature detersiveperformance. As such, they are especially desirable as ingredients ofvarious household and/or personal care cleaning/treatment compositionswherein low washing temperatures are preferred.

As used herein, “fraction of modern carbon” or “f_(M)” has the samemeaning as defined by National Institute of Standards and Technology(NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known asoxalic acids standards HOxI and HOxII, respectively. The fundamentaldefinition relates to 0.95 times the ¹⁴C/¹²C isotope ratio HoxI(referenced to AD 1950). This is roughly equivalent to decay-correctedpre-Industrial Revolution wood. For the current living biosphere (plantmaterial), f_(M) is approximately 1.1.

“Gene knockout”, as used herein, refers to a procedure by which a geneencoding a target protein is modified or inactivated so as to reduce oreliminate the function of the intact protein. Inactivation of the genemay be performed by general methods such as mutagenesis by UVirradiation or treatment with N-methyl-N′-nitro-N-nitrosoguanidine,site-directed mutagenesis, homologous recombination, insertion-deletionmutagenesis, or “Red-driven integration” (Datsenko et al., Proc. Natl.Acad. Sci. USA, 97: 6640-45 (2000)). For example, in one embodiment, aconstruct is introduced into a host cell, such that it is possible toselect for homologous recombination events in the host cell. One ofskill in the art can readily design a knock-out construct including bothpositive and negative selection genes for efficiently selectingtransfected cells that undergo a homologous recombination event with theconstruct. The alteration in the host cell may be obtained, for example,by replacing through a single or double crossover recombination a wildtype DNA sequence by a DNA sequence containing the alteration. Forconvenient selection of transformants, the alteration may, for example,be a DNA sequence encoding an antibiotic resistance marker or a genecomplementing a possible auxotrophy of the host cell. Mutations include,but are not limited to, deletion-insertion mutations. An example of suchan alteration includes a gene disruption (i.e., a perturbation of agene) such that the product that is normally produced from this gene isnot produced in a functional form. This could be due to a completedeletion, a deletion and insertion of a selective marker, an insertionof a selective marker, a frameshift mutation, an in-frame deletion, or apoint mutation that leads to premature termination. In some instances,the entire mRNA for the gene is absent. In other situations, the amountof mRNA produced varies.

Calculations of “homology” between two sequences can be performed asfollows. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence that isaligned for comparison purposes is at least about 30%, preferably atleast about 40%, more preferably at least about 50%, even morepreferably at least about 60%, and even more preferably at least about70%, at least about 80%, at least about 90%, or about 100% of the lengthof the reference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein, amino acid or nucleic acid “identity” is equivalent toamino acid or nucleic acid “homology”). The percent identity between thetwo sequences is a function of the number of identical positions sharedby the sequences, taking into account the number of gaps and the lengthof each gap, which need to be introduced for optimal alignment of thetwo sequences.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent homology between twoamino acid sequences is determined using the Needleman and Wunsch(1970), J. Mol. Biol. 48:444 453, algorithm that has been incorporatedinto the GAP program in the GCG software package, using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent homology between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage, using a NWSgapdna. CMP matrix and a gap weight of about 40, 50,60, 70, or 80 and a length weight of about 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused if the practitioner is uncertain about which parameters should beapplied to determine if a molecule is within a homology limitation ofthe claims) are a Blossum 62 scoring matrix with a gap penalty of 12, agap extend penalty of 4, and a frameshift gap penalty of 5.

Other methods for aligning sequences for comparison are well known inthe art. Various programs and alignment algorithms are described in, forexample, Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Pearson &Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene73:237 244, 1988; Higgins & Sharp, CABIOS 5:151-153, 1989; Corpet etal., Nucleic Acids Research 16:10881-10890, 1988; Huang et al., CABIOS8:155-165, 1992; and Pearson et al., Methods in Molecular Biology24:307-331, 1994. and Altschul et al., J. Mol. Biol. 215:403-410, 1990.

As used herein, a “host cell” is a cell used to produce a productdescribed herein (e.g., a branched fatty alcohol described herein). Ahost cell can be modified to express or overexpress selected genes or tohave attenuated expression of selected genes. Non-limiting examples ofhost cells include plant, animal, human, bacteria, yeast, or filamentousfungi cells.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found, for example, in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. Aqueous and nonaqueous methods are described in thatreference, and either method can be used. An example of hybridizationconditions referred to herein are as follows: 1) low stringencyhybridization conditions in 6× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at50° C. (the temperature of the washes can be increased to 55° C. for lowstringency conditions); 2) medium stringency hybridization conditions in6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC atabout 45° C., followed by one or more washes in 0.2.X SSC, 0.1% SDS at65° C.; and 4) very high stringency hybridization conditions in 0.5Msodium phosphate, 7% SDS at 65° C., followed by one or more washes at0.2×SSC, 1% SDS at 65° C. Very high stringency conditions of 4) are thepreferred conditions unless otherwise specified.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs or RNAs,respectively, that are present in the natural source of the nucleicacid. Moreover, by an “isolated nucleic acid” is meant to includenucleic acid fragments, which are not naturally occurring as fragmentsand would not be found in the natural state. The term “isolated” is alsoused herein to refer to polypeptides, which are isolated from othercellular proteins, and is meant to encompass both purified andrecombinant polypeptides. The term “isolated” as used herein also refersto a nucleic acid or peptide that is substantially free of cellularmaterial, viral material, or culture medium when produced by recombinantDNA techniques. The term “isolated” as used herein also refers to anucleic acid or peptide that is substantially free of chemicalprecursors or other chemicals when chemically synthesized.

As used herein, the “level of expression of a gene in a cell” refers tothe level of mRNA, pre-mRNA nascent transcript(s), transcript processingintermediates, mature mRNA(s), and degradation products encoded by thegene in the cell.

As used herein, the term “microorganism” means prokaryotic andeukaryotic microbial species from the domains Archaea, Bacteria, andEucarya, the latter including yeast and filamentous fungi, protozoa,algae, or higher Protista. The terms “microbial cells” (i.e., cells frommicrobes) and “microbes” are used interchangeably and refer to cells orsmall organisms that can only be seen with the aid of a microscope.

As used herein, the term “nucleic acid” refers to polynucleotides, suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides, ESTs, chromosomes,cDNAs, mRNAs, and rRNAs.

As used herein, the term “operably linked” means that selectednucleotide sequence (e.g., encoding a polypeptide described herein) isin proximity to a promoter to allow the promoter to regulate expressionof the selected DNA. In addition, the promoter is located upstream ofthe selected nucleotide sequence in terms of the direction oftranscription and translation. By “operably linked” is meant that anucleotide sequence and a regulatory sequence(s) are connected in such away as to permit gene expression when the appropriate molecules (e.g.,transcriptional activator proteins) are bound to the regulatorysequence(s).

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

As used herein, “overexpress” means to express or cause to be expresseda nucleic acid or polypeptide in a cell at a greater concentration thanis normally expressed in a corresponding wild-type cell. For example, apolypeptide can be “overexpressed” in a recombinant host cell when thepolypeptide is present in a greater concentration in the recombinanthost cell compared to its concentration in a non-recombinant host cellof the same species.

As used herein, “partition coefficient” or “P” is defined as theequilibrium concentration of a compound in an organic phase divided bythe concentration at equilibrium in an aqueous phase (e.g., fermentationbroth). In one embodiment of a bi-phasic system described herein, theorganic phase is formed by the fatty aldehyde or fatty alcohol duringthe production process. However, in some examples, an organic phase canbe provided, such as by providing a layer of octane, to facilitateproduct separation. When describing a two phase system, the partitioncharacteristics of a compound can be described as logP. For example, acompound with a logP of 1 would partition 10:1 to the organic phase:aqueous phase. A compound with a logP of −1 would partition 1:10 to theorganic phase: aqueous phase. By choosing an appropriate fermentationbroth and organic phase, a branched fatty aldehyde or branched fattyalcohol with a high logP value can separate into the organic phase evenat very low concentrations in the fermentation vessel.

As used herein, the term “purify,” “purified,” or “purification” meansthe removal or isolation of a molecule from its environment by, forexample, isolation or separation. “Substantially purified” molecules areat least about 60% free, preferably at least about 75% free, and morepreferably at least about 90% free from other components with which theyare associated. As used herein, these terms also refer to the removal ofcontaminants from a sample. For example, the removal of contaminants canresult in an increase in the percentage of branched fatty aldehyde orbranched fatty alcohol in a sample. For example, when branched fattyalcohols are produced in a host cell, the branched fatty alcohols can bepurified by the removal of host cell proteins, or by simply separatingand removing linear fatty alcohols that are produced during the sameprocess. After purification, the percentage of branched fatty alcoholsin the sample is increased.

The terms “purify,” “purified,” and “purification” do not requireabsolute purity. They are relative terms. Thus, for example, whenbranched fatty alcohols are produced in host cells, a purified branchedfatty alcohol is one that is substantially separated from other cellularcomponents (e.g., nucleic acids, polypeptides, lipids, carbohydrates, orother compounds, such as, for example, linear fatty alcohols). Inanother example, a purified branched fatty alcohol preparation is one inwhich the branched fatty alcohol is substantially free fromcontaminants, such as those that might be present followingfermentation. In some embodiments, a branched fatty alcohol is purifiedwhen at least about 50% by weight of a sample is composed of thebranched fatty alcohol. In other embodiments, a branched fatty alcoholis purified when at least about 60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%,or 99% or more by weight of a sample is composed of the branched fattyalcohol.

As used herein, the term “recombinant polypeptide” refers to apolypeptide that is produced by recombinant DNA techniques, whereingenerally DNA encoding the expressed protein or RNA is transferred intoa suitable expression vector and that is in turn used to transform ahost cell to produce the polypeptide or RNA.

As used herein, the term “substantially identical” (or “substantiallyhomologous”) is used to refer to a first amino acid or nucleotidesequence that contains a sufficient number of identical or equivalent(e.g., with a similar side chain, such as involving conservative aminoacid substitutions) amino acid residues or nucleotides to a second aminoacid or nucleotide sequence such that the first and second amino acid ornucleotide sequences have similar activities.

As used herein, the term “surfactants” refers broadly to surface activeagents. These agents are typically amphipathic molecules comprising bothhydrophilic and hydrophobic moieties that partition preferentially atthe interface between fluid phases with different degrees of polarityand hydrogen bonding, such as, for example, an oil/water interface, oran air/water interface. Surfactants are capable of reducing surface andinterfacial tension and forming microemulsions. These characteristicsconfer detergency, emulsifying, foaming and dispersing traits, makingthem some of the most versatile process chemicals.

Surfactants can be natural or synthetic in origin. Surfactants fromnatural origin can be derived from, for example, vegetable or animalsources. Surfactants derived from synthetic origin are typically thosederived from petroleum.

There are many types of surfactants, including, for example, anionicsurfactants, cationic surfactants, non-ionic surfactants, andamphoteric/zwitterionic surfactants, each with distinct characteristics.

The hydrophobic end of an anionic surfactant is negatively charged insolution. As a result, they have good cleaning properties and highsudding potentials, which make them particularly effective as some ofthe most widely used types of surfactants in, for example, laundrydetergents, dishwashing liquids, and shampoos. Known anionic surfactantsinclude, for example, alkyl sulfates, alkyl ethoxylate sulfates, andsoaps.

The hydrophobic end of a cationic surfactant is positively charged insolution. Three types of cationic surfactants are the most commonlyknown. The first type is the esterquat, which is widely included in, forexample, fabric treatment agents or softeners and in detergents withbuilt-in softeners. This is because esterquat is capable of addingsoftness to fabrics. The second type is a mono alkyl quaternary system,which is found in many household cleaners due to its disinfecting and/orsanitizing properties.

Non-ionic surfactants do not have an electrical charge in solution,making them resistant to water hardness deactivation. They are typicallyexcellent grease removers. The most commonly used non-ionic surfactantsare ethers or derivatives of fatty alcohols.

Amphoteric/zwitterionic surfactants are milder than the other types ofsurfactants, making them particularly suitable for use in personal orbeauty care cleaning/treatment products. They may contain twooppositely-charged groups. While the positive charge is typicallyconferred by ammonium, the source of the negative charge can vary. Forexample, the negative charge can be conferred by carboxylate, sulfate,sulfonate, or a combination thereof. They can be anionic (e.g.,negatively charged), cationic (e.g., positively charged) or non-ionic(e.g., no charge) in solution, depending on the acidity or pH of thesolution. They have good compatibility with the other types ofsurfactants and are well known for being soluble and effective in thepresence of high concentrations of electrolytes, acids and alkalis. Anexample of an amphoteric/zwitterionic surfactant is an alkyl betaine.

In typical applications, different types of surfactants are blended orotherwise used together to achieve an array of desirable properties.

As used herein, the term “synthase” means an enzyme that catalyzes asynthesis process. As used herein, the term synthase includes synthases,synthetases, and ligases.

As used herein, the term “transfection” means the introduction of anucleic acid (e.g., via an expression vector) into a recipient cell bynucleic acid-mediated gene transfer.

As used herein, “transformation” refers to a process in which a cell'sgenotype is changed as a result of the cellular uptake of exogenous DNAor RNA. This may result in the transformed cell expressing a recombinantform of an RNA or polypeptide. In the case of antisense expression fromthe transferred gene, the expression of a naturally-occurring form ofthe polypeptide is disrupted.

As used herein, a “transport protein” is a polypeptide that facilitatesthe movement of one or more compounds in and/or out of a cellularorganelle and/or a cell.

As used herein, a “variant” of polypeptide X refers to a polypeptidehaving the amino acid sequence of peptide X in which one or more aminoacid residues is altered. The variant may have conservative changes ornonconservative changes. Guidance in determining which amino acidresidues may be substituted, inserted, or deleted without affectingbiological activity may be found using computer programs well known inthe art, for example, LASERGENE software (DNASTAR).

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to that of agene or the coding sequence thereof. This definition may also include,for example, “allelic,” “splice,” “species,” or “polymorphic” variants.A splice variant may have significant identity to a referencepolynucleotide, but will generally have a greater or fewer number ofpolynucleotides due to alternative splicing of exons during mRNAprocessing. The corresponding polypeptide may possess additionalfunctional domains or an absence of domains. Species variants arepolynucleotide sequences that vary from one species to another. Theresulting polypeptides generally will have significant amino acidsequence identity relative to each other. A polymorphic variant is avariation in the polynucleotide sequence of a particular gene betweenindividuals of a given species.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of useful vector is an episome (i.e., a nucleic acidcapable of extra-chromosomal replication). Useful vectors are thosecapable of autonomous replication and/or expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids,” whichrefer generally to circular double stranded DNA loops that, in theirvector form, are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably, as theplasmid is the most commonly used form of vector. However, also includedare such other forms of expression vectors that serve equivalentfunctions and that become known in the art subsequently hereto.

Surfactants and Cleaning Compositions Comprising a Microbially ProducedBranched Fatty Alcohol or a Branched Fatty Alcohol Derivative Thereof

The invention provides a surfactant composition comprising one or moremicrobially produced branched chain fatty alcohols and/or derivativesthereof. The invention further provides a detergent/cleaningcomposition, such as, for example, a household cleaning composition or apersonal or beauty care cleaning composition, comprising such asurfactant.

In one aspect, the invention features a surfactant compositioncomprising branched chain fatty alcohols and/or derivatives thereofproduced by microbes. In some embodiments, the microbially producedbranched fatty alcohol and/or derivative thereof is produced by a hostcell expressing genes encoding at least one subunit of a branched-chainalpha-keto acid dehydrogenase polypeptide. The host cell expresses genesencoding at least two subunits of a branched-chain alpha-keto aciddehydrogenase polypeptide. For example, the host cell expresses a set ofgenes encoding the first subunit and a second subunit of abranched-chain alpha-keto acid dehydrogenase polypeptide. In certainembodiments, the host cell expresses a third gene encoding the secondsubunit of a branched-chain alpha-keto acid dehydrogenase polypeptide.In some embodiments, the first and second polypeptides havebranched-chain alpha-keto acid decarboxylase activity, and the thirdpolypeptide has lipoamide acyltransferase activity. In furtherembodiments, the host cell expresses a fourth gene encoding the thirdsubunit of a branched-chain alpha-keto acid dehydrogenase polypeptide.In some embodiments, the fourth polypeptide has lipoamide dehydrogenaseactivity.

In some embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell expressing a geneencoding a beta ketoacyl-ACP synthase polypeptide. In certainembodiments, the beta ketoacyl-ACP synthase polypeptide has FabHactivity. In certain embodiments the beta ketoacyl-ACP synthase hasspecificity for branched-chain acyl-CoA substrates.

In some embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell expressing a set ofgenes encoding at least one subunit of a branched-chain alpha-keto aciddehydrogenase complex. Specifically, the microbially produced branchedfatty alcohol and/or derivative thereof is produced by a host cellexpressing a first gene encoding a first polypeptide comprising theamino acid sequence that is any one of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13,and 15, or one that has at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99% sequence identity to the aminoacid sequence of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15,or a variant thereof; a second gene encoding a second polypeptidecomprising an amino acid sequence of any one of SEQ ID NOs:24, 26, 28,30, 32, 34, 36, and 38, or one that has at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99% sequenceidentity to the amino acid sequence of any one of SEQ ID NOs:24, 26, 28,30, 32, 34, 36, and 38, or a variant thereof. In certain embodiments,the host cell also expresses a third gene encoding a third polypeptidecomprising the amino acid sequence of any one of SEQ ID NOs:47, 49, 51,53, 55, 57, 59, and 61, or one that has at least 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99% sequenceidentity to the amino acid sequence of any one of SEQ ID NOs:47, 49, 51,53, 55, 57, 59, and 61, or a variant thereof. In some embodiments, thebranched fatty aldehyde, branched fatty alcohol, or a derivative thereofis isolated from the host cell, for example, isolated from theextracellular environment of the host cell. In some embodiments, thebranched fatty aldehyde, branched fatty alcohol, or the derivativethereof is spontaneously secreted, completely or partially, from thehost cell. In alternative embodiments, the branched fatty aldehyde,branched fatty alcohol, or the derivative thereof is transported intothe extracellular environment. In further embodiments, the branchedfatty aldehyde, branched fatty alcohol, or the derivative thereof ispassively transported or spontaneously secreted into the extracellularenvironment.

The first polypeptide comprises the amino acid sequence of any one ofSEQ ID NOs:1, 3, 5, 7, 9, 11, 13, and 15, with one or more amino acidsubstitutions, additions, insertions, or deletions, the secondpolypeptide comprises the amino acid sequence of any one of SEQ ID NOs:24, 26, 28, 30, 32, 34, 36, and 38, wherein the first and secondpolypeptides together have alpha-keto acid decarboxylase activity. Incertain embodiments, the first polypeptide comprises one or more or allof the amino acid sequence motifs selected from SEQ ID NOs:17-23. Thesecond polypeptide comprises one or more or all of the amino acidsequence motifs selected from SEQ ID NOs:40-46. In some embodiments, thethird polypeptide comprises an amino acid sequence of any one of SEQ IDNOs: 47, 49, 51, 53, 55, 57, 59, and 61, with one or more amino acidsubstitutions, additions, insertions, or deletions, wherein the thirdpolypeptide has lipoamide acyltransferase activity. The thirdpolypeptide comprises one or more or all of the amino acid sequencemotifs selected from SEQ ID NOs:63-68. In some embodiments, the first,second and third polypeptides are capable of catalyzing the conversionof alpha-keto acids to branched acyl-CoAs. It is within the capacity ofthose skilled in the art to devise a suitable enzymatic assay using theappropriate substrates. Examples of such assays are described herein.

In some embodiments, the first, second, and third polypeptidesindependently comprises 1 or more, 5 or more, 10 or more, 15 or more, 20or more, 30 or more, 40 or more, 50 or more, or 100 or more of thefollowing conservative amino acid substitutions: replacement of analiphatic amino acid, such as alanine, valine, leucine, and isoleucine,with another aliphatic amino acid; replacement of a serine with athreonine; replacement of a threonine with a serine; replacement of anacidic residue, such as aspartic acid and glutamic acid, with anotheracidic residue; replacement of a residue bearing an amide group, such asasparagine and glutamine, with another residue bearing an amide group;exchange of a basic residue, such as lysine and arginine, with anotherbasic residue; and replacement of an aromatic residue, such asphenylalanine and tyrosine, with another aromatic residue. In someembodiments, the first and second polypeptides independently comprisesabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, or more amino acid substitutions, additions, insertions, ordeletions. In some embodiments, the third polypeptide comprises about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, or more amino acid substitutions, additions, insertions, ordeletions. In some embodiments, the first and second polypeptides havebranched-chain alpha-keto acid decarboxylase activity and the thirdpolypeptide has lipoamide acyltransferase activity. In some embodiments,the first, second and third polypeptides are capable of catalyzing theconversion of branched alpha-keto acids to branched acyl-CoAs.

In certain embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell expressing a fourthgene encoding a fourth polypeptide comprising the amino acid sequence ofany one of SEQ ID NOs:69, 71, 73, 75, 77, 79, 81, and 83, or one thathas at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99% sequence identity to the amino acid sequence of anyone of SEQ ID NOs:69, 71, 73, 75, 77, 79, 81, and 83, or a variantthereof. In some embodiments, the branched fatty aldehyde, branchedfatty alcohol, or a derivative thereof is isolated from the host cell,for example, from the extracellular environment. In certain embodiments,the branched fatty aldehyde, branched fatty alcohol, or the derivativethereof is spontaneously secreted, partially or completely, into theextracellular environment. In other embodiments, the branched fattyaldehyde, branched fatty alcohol, or the derivative thereof istransported into the extracellular environment. In certain embodiments,the branched fatty aldehyde, branched fatty alcohol or the derivativethereof is passively transported into the extracellular environment.

The fourth polypeptide comprises the amino acid sequence of any one ofSEQ ID NOs:69, 71, 73, 75, 77, 79, 81, and 83, with one or more aminoacid substitutions, additions, insertions, or deletions, and thepolypeptide has lipoamide dehydrogenase activity. In certainembodiments, the fourth polypeptide comprises one or more or all ofamino acid sequence motifs selected from SEQ ID NOs:85-89. In someembodiments, the fourth polypeptide comprises 1 or more, 5 or more, 10or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, or100 or more of the following conservative amino acid substitutions:replacement of an aliphatic amino acid, such as alanine, valine,leucine, and isoleucine, with another aliphatic amino acid; replacementof a serine with a threonine; replacement of a threonine with a serine;replacement of an acidic residue, such as aspartic acid and glutamicacid, with another acidic residue; replacement of a residue bearing anamide group, such as asparagine and glutamine, with another residuebearing an amide group; exchange of a basic residue, such as lysine andarginine, with another basic residue; and replacement of an aromaticresidue, such as phenylalanine and tyrosine, with another aromaticresidue. In some embodiments, the fourth polypeptide comprises about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, or more amino acid substitutions, additions, insertions, ordeletions. In some embodiments, the fourth polypeptide has lipoamidedehydrogenase activity. In some embodiments, the first, second, thirdand fourth polypeptides have branched chain alpha-keto aciddecarboxylase and/or lipoamide acyltransferase and/or lipoamidedehydrogenase activity. In some embodiments, the first, second, thirdand fourth polypeptides, optionally forming a complex, are capable ofcatalyzing the conversion alpha-keto acids to branched-chain acyl-CoAs.

In certain embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell further expressinga gene encoding a beta-ketoacyl ACP synthase comprising the amino acidsequence of any one of SEQ ID NOs: 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, and 120, or one that has at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99% sequence identity to the amino acid sequence of any one of SEQID NOs:90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, and 120, or a variant thereof. In some embodiments, the branchedfatty aldehyde, branched fatty alcohol, or a derivative thereof isisolated from the host cell, for example, from the extracellularenvironment. In certain embodiments, the branched fatty aldehyde,branched fatty alcohol, or the derivative thereof is spontaneouslysecreted, partially or completely, into the extracellular environment.In other embodiments, the branched fatty aldehyde, branched fattyalcohol, or the derivative thereof is transported into the extracellularenvironment. In certain embodiments, the branched fatty aldehyde,branched fatty alcohol, or the derivative thereof is passivelytransported into the extracellular environment.

The beta ketoacyl-ACP synthase polypeptide comprises the amino acidsequence of any one of SEQ ID NOs:90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, and 120, with one or more amino acidsubstitutions, additions, insertions, or deletions. In some embodiments,the beta ketoacyl-ACP synthase polypeptide comprises one or more or allof amino acid sequence motifs selected from SEQ ID NOs:122-127. In someembodiments, the polypeptide has FabH activity. In certain embodiments,the beta ketoacyl-ACP synthase polypeptide has specificity forbranched-chain fatty acyl-CoA substrates. In certain embodiments, thepolypeptide is capable of catalyzing the condensation reaction between abranched acyl-CoA and malonyl-ACP. It is within the capacity of thoseskilled in the art to devise a suitable enzymatic assay using theappropriate substrates in order to distinguish those polypeptides havingsequence homology to the beta-ketoacyl-ACP synthase polypeptides hereinbut are not suitable or does not have specificity for branched-chainsubstrates. Two examples of such enzymatic assays are described herein.

The beta ketoacyl-ACP synthase polypeptide can comprise 1 or more, 5 ormore, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 ormore, or 100 or more of the following conservative amino acidsubstitutions: replacement of an aliphatic amino acid, such as alanine,valine, leucine, and isoleucine, with another aliphatic amino acid;replacement of a serine with a threonine; replacement of a threoninewith a serine; replacement of an acidic residue, such as aspartic acidand glutamic acid, with another acidic residue; replacement of a residuebearing an amide group, such as asparagine and glutamine, with anotherresidue bearing an amide group; exchange of a basic residue, such aslysine and arginine, with another basic residue; and replacement of anaromatic residue, such as phenylalanine and tyrosine, with anotheraromatic residue. In some embodiments, the polypeptide comprises about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100,200 or more amino acid substitutions, additions, insertions, ordeletions. In some embodiments, the polypeptide has FabH activity. Insome embodiments, the polypeptide has specificity for branched-chainacyl-CoAs. In some embodiments, the polypeptide is capable of catalyzingthe condensation of a branched acyl-CoA and malonyl-ACP.

In certain embodiments, the first polypeptide comprises an amino acidsequence motif of any one of or one or more or all of SEQ ID NOs:17-23,wherein the first polypeptide is of about 200 to about 800 amino acidresidues in length, or about 300 to about 700 amino acid residues inlength, or about 400 to about 600 amino acids in length. In someembodiments, the second polypeptide comprises an amino acid sequencemotif of any one of or one or more or all of SEQ ID NOs:40-46, whereinthe second polypeptide is about 200 to about 800 amino acid residues inlength, or about 300 to about 700 amino acid residues in length, orabout 400 to about 600 amino acid residues in length. In someembodiments, the third polypeptide comprises an amino acid sequencemotif of any one of or one or more or all of SEQ ID NOs:63-68, whereinthe first polypeptide is of about 200 to about 800 amino acid residuesin length, or about 300 to about 700 amino acid residues in length, orabout 400 to about 600 amino acid residues in length. In someembodiments, the first, second and optionally the third polypeptides arecapable of catalyzing the conversion of alpha-keto acid substrates tobranched acyl-CoAs.

In certain embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell further expressinga gene encoding a fatty aldehyde biosynthesis polypeptide selected fromthose listed in the Table 6, or a variant thereof. In some embodiments,the fatty aldehyde biosynthesis polypeptide comprises the amino acidsequence of an enzyme listed in Table 6, with one or more amino acidsubstitutions, additions, insertions, or deletions, and the polypeptidehas carboxylic acid reductase activity. In some embodiments, thepolypeptide has fatty acid reductase activity. In some embodiments, thefatty aldehyde biosynthesis polypeptide comprises one or more of thefollowing conservative amino acid substitutions. In some embodiments,the fatty aldehyde biosynthesis polypeptide has about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acidsubstitutions, additions, insertions, or deletions. In some embodiments,the polypeptide has carboxylic acid reductase activity. In someembodiments, the polypeptide has fatty acid reductase activity.

In some embodiments, the branched fatty alcohol or a derivative thereofis isolated from the host cell, for example, from the extracellularenvironment. In some embodiments, the branched fatty alcohol or thederivative thereof is spontaneously secreted, partially or completely,from the host cell. In alternative embodiments, the branched fattyalcohol or the derivative thereof is transported into the extracellularenvironment. In other embodiments, the branched fatty alcohol or thederivative thereof is passively transported into the extracellularenvironment.

In some embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell wherein a geneencoding a fatty acid synthase is modified. For example, modifying theexpression of a gene encoding a fatty acid synthase includes expressinga gene encoding a fatty acid synthase in the host cell and/or increasingthe expression or activity of an endogenous fatty acid synthase in thehost cell. Alternatively, modifying the expression of a gene encoding afatty acid synthase includes attenuating a gene encoding a fatty acidsynthase in the host cell and/or decreasing the expression or activityof an endogenous fatty acid synthase in the host cell. In someembodiments, the fatty acid synthase is a thioesterase. In particularembodiments, the thioesterase is encoded by tesA, tesA without leadersequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.

In some embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell expressing a geneencoding a fatty alcohol biosynthesis polypeptide. The fatty alcoholbiosynthesis polypeptide is, for example, an alcohol dehydrogenase. Inparticular embodiments, the fatty alcohol biosynthesis polypeptide isone selected from the enzymes listed in Table 8, or a variant thereof.

In certain other embodiments, the microbially produced branched fattyalcohol and/or derivative thereof is produced by a host cell expressinga gene encoding another aldehyde biosynthetic polypeptide or an acyl-ACPreductase polypeptide comprising the amino acid sequence of any of theenzymes listed in Table 7, or a variant thereof. In some embodiments,the branched fatty alcohol or derivative thereof is isolated from thehost cell, for example, from the extracellular environment. In certainembodiments, the branched fatty alcohol or derivative thereof isspontaneously secreted, partially or completely, from the host cell. Inalternative embodiments, the branched fatty alcohol or derivativethereof is transported into the extracellular environment. In otherembodiments, the branched fatty alcohol or derivative thereof ispassively transported into the extracellular environment.

The acyl-ACP reductase polypeptide, for example, comprises the aminoacid sequence of an enzyme selected from those listed in Table 7, withone or more amino acid substitutions, additions, insertions, ordeletions, and the polypeptide has reductase activity. In certainembodiments, the polypeptide is capable of catalyzing the conversion ofa suitable biological substrate into an aldehyde. The acyl-ACP reductasepolypeptide, for example, comprises one or more conservative amino acidsubstitutions, or has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, or more amino acid substitutions,additions, insertions, or deletions. In some embodiments, thepolypeptide has reductase activity. In some embodiments, the polypeptideis capable of catalyzing the conversion of a suitable biologicalsubstrate into an aldehyde.

In any of the above-described embodiments, the microbially producedbranched fatty alcohol and/or derivative thereof is produced by a hostcell, which is genetically engineered to express an attenuated level ofa fatty acid degradation enzyme relative to a wild type host cell. Forexample, the host cell is genetically engineered to express anattenuated level of an acyl-CoA synthase relative to a wild type hostcell. In particular embodiments, the host cell expresses an attenuatedlevel of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL,Pfl-4354, EAV15023, fadD1, fadD2, RPC_4074, fadDD35, fadDD22, faa3p orthe gene encoding the protein ZP_01644857. In certain embodiments, thegenetically engineered host cell comprises a knockout of one or moregenes encoding a fatty acid degradation enzyme, such as theaforementioned acyl-CoA synthase genes. In certain embodiments, the hostcell is genetically engineered to express, relative to a wild type hostcell, a decreased level of at least one of a gene encoding an acyl-CoAdehydrogenase, a gene encoding an outer membrane protein receptor, and agene encoding a transcriptional regulator of fatty acid biosynthesis. Insome embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE.In some embodiments, the gene encoding an outer membrane proteinreceptor is tonA (also known as fhuA). Yet in other embodiments, thegene encoding a transcriptional regulator of fatty acid biosynthesis isfabR.

In yet other embodiments, the microbially produced branched fattyalcohol and/or derivative thereof is produced by a host cell, which isgenetically engineered to express an attenuated level of adehydratase/isomerase enzyme, such as an enzyme encoded by fabA or by agene listed in Table 1 or Table 2. In some embodiments, the host cellcomprises a knockout of fabA or a gene listed in Table 1 or Table 2. Inother embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell, which isgenetically engineered to express an attenuated level of an endogenousketoacyl-ACP synthase, such as an enzyme encoded by fabB or by a genelisted in Table 3 or Table 4. In certain embodiments, the host cellcomprises a knockout of fabB or a gene listed in Table 3 or Table 4. Inyet other embodiments, the host cell is genetically engineered toexpress a modified level of a gene encoding a desaturase enzyme, such asdesA.

In certain embodiments, the branched-chain alpha-keto acid dehydrogenasecomplex polypeptides, the beta ketoacyl-ACP synthase polypeptide, thealdehyde biosynthesis polypeptide, the fatty acid synthase, the acyl-ACPreductase, the alcohol biosynthesis polypeptide, and the fatty aciddegradation enzyme polypeptide are each independently obtained from abacterium, a plant, an insect, a yeast, a fungus, or a mammal. Forexample, each of the above-mentioned polypeptides is from a mammaliancell, plant cell, insect cell, yeast cell, fungus cell, filamentousfungi cell, bacterial cell, or any other organism described herein. Incertain embodiments, the branched-chain alpha-keto acid dehydrogenasecomplex polypeptides can be from a bacterium that uses branched aminoacids as carbon source, including, for example, Pseudomonas putida or aBacillus subtilis. In certain embodiments, the branched-chain alpha-ketoacid dehydrogenase complex polypeptide can be from a bacterium thatcomprises branched fatty acids in its phospholipids, including, forexample, a Legionella, Stenotrophomonas, Alteromonas, Flavobacterium,Myxococcus, Bacteroides, Micrococcus, Staphylococcus, Bacillus,Clostridium, Listeria, Lactococcus, or Streptomyces bacterium. In someembodiments, the bacterium is a Leginella pneumophila, Stenotrophomonasmaltophilia, Alteromonas macleodii, Flabobacterium phsychrophilum,Myxococcus Xanthus, Bacteroides thetaiotaomicron, Macrococcus luteus,Staphylococcus aureus, Clostridium thermocellum, Listeria monocytogenes,Streptomyces lividans, Streptomyces coelicolor, Streptomycesglaucescens, Streptococcus pneumoniae, Streptomyces peucetius,Streptococcus pyogenes, Escherichia coli, Escherichia coli K-12,Lactococcus lactis ssp. Lactis, Mycobacterium tuberculosis, Enterococcustuberculosis, Bacillus subtilis, Lactobacillus plantarum. In certainembodiments, suitable fatty aldehyde biosynthesis polypeptides, fattyalcohol biosynthesis polypeptides, acyl-ACP reductases, and otherpolypeptides of the invention can be from a mycobacterium selected fromthe group consisting of Mycobacterium smegmatis, Mycobacteriumabscessus, Mycobacterium avium, Mycobacterium bovis, Mycobacteriumtuberculosis, Mycobacterium leprae, Mycobacterium marinum, andMycobacterium ulcerans. In other embodiments, the bacterium is Nocardiasp. NRRL 5646, Nocardia farcinica, Streptomyces griseus, Salinisporaarenicola, or Clavibacter michiganenesis. In certain furtherembodiments, the polypeptide of the invention is derived from acyanobacterium, including, for example, Synechococcus elongatus PCC7942,Synechocystis sp. PCC6803, Cyanothece sp. ATCC51142, Prochlorococcusmarinus subsp. pastoris str. CCMP1986 PMM0533, Gloeobacter violaceusPCC7421, Nostoc punctiforme PCC73102, Anabaena variabilis ATCC29413,Synechococcus elongatus PCC6301, and Nostoc sp. PCC 7120, Microcoleuschthonoplastes PCC7420, Arthrospira maxima CS-328, Lyngbya sp. PCC8106,Nodularia spumigena CCY9414, Trichodesmium erythraeum IMS101,Microcystis aeruginosa, Nostoc azollae, Anabaena variabilis, Crocophaerawatsonii, Thermosynechococcus elongatus, Gloeobacer violaceus,Cyanobium, or Prochlorococcus marinus.

In some embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell cultured in thepresence of at least one biological substrate for the branched-chainalpha-keto acid dehydrogenase polypeptides, the beta ketoacyl-ACPsynthase polypeptide, the aldehyde biosynthesis polypeptide, theacyl-ACP reductase, and/or the alcohol biosynthesis polypeptide.Suitable substrate for the branched-chain alpha-keto acid dehydrogenasepolypeptides can include, without limitation, 2-oxo-isovalerate,2-oxo-isobutyrate, or 2-oxo-3-methyl-valerate.

In another aspect, the invention features a surfactant or detergentcomposition comprising a microbially produced branched fatty alcohol ora derivative thereof. In some embodiments, the microbially producedbranched fatty alcohol and/or derivative thereof is produced by a hostcell expressing a first polynucleotide that hybridizes to a complementof a nucleotide sequence of any one of SEQ ID NOs:2, 4, 6, 8, 10, 12,14, and 16, or to a fragment thereof, and a second polynucleotide thathybridizes to a complement of a second polynucleotide sequence of anyone of SEQ ID NOs:25, 27, 29, 31, 33, 35, 37, and 39. In certainembodiments, the microbially produced branched fatty alcohol and/orderivative thereof is produced by a host cell expressing a thirdpolynucleotide that hybridizes to a complement of a third nucleotidesequence of any one of SEQ ID NOs:48, 50, 52, 54, 56, 58, 60, and 62, orto a fragment thereof, wherein the first and second polynucleotidesencode the first and second polypeptides having branched-chainalpha-keto acid decarboxylase activity, and wherein the thirdpolynucleotide encodes a polypeptide having lipoamide acyltransferaseactivity. In some embodiments, the first and the second polypeptides,optionally forming a single subunit, optionally together with the thirdpolypeptide, are capable of catalyzing the conversion of branched-chainalpha-keto acids to branched acyl-CoAs.

The first polynucleotide hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions, to acomplement of the nucleotide sequence of any one of SEQ ID NOs:2, 4, 6,8, 10, 12, 14, and 16, or to a fragment thereof. The secondpolynucleotide hybridizes under low stringency, medium stringency, highstringency, or very high stringency conditions, to a complement of thenucleotide sequence of any one of SEQ ID NOs: 25, 27, 29, 31, 33, 35,37, and 39, or to a fragment thereof. The third polynucleotidehybridizes under low stringency, medium stringency, high stringency, orvery high stringency conditions, to a complement of the nucleotidesequence of any one of SEQ ID NOs: 48, 50, 52, 54, 56, 58, 60, and 62.

In some embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell expressing a fourthpolynucleotide that hybridizes to a complement of a nucleotide sequenceof any one of SEQ ID NOs:70, 72, 74, 76, 78, 80, 82, and 84, or to afragment thereof, wherein the fourth polynucleotide encodes apolypeptide having lipoamide dehydrogenase activity. In someembodiments, the first, second, and optionally the third and/or fourthpolypeptides are capable of catalyzing the conversion of branched-chainalpha-keto acids into branched acetyl-CoAs.

In some embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell expressing apolynucleotide that hybridizes to a complement of a nucleotide sequenceof any one of SEQ ID NOs:91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, and 121, or to a fragment thereof, wherein thepolynucleotide encodes a polypeptide having beta ketoacyl-ACP synthaseactivity. In some embodiments, the polypeptide is capable of catalyzingthe condensation of a branched acyl-CoA with malonyl-ACP. In someembodiments, the polypeptide has FabH activity. In some embodiments, thepolypeptide has specificity for branched acyl-CoA substrates. Thepolynucleotide hybridizes under low stringency, medium stringency, highstringency, or very high stringency conditions, to a complement of thenucleotide sequence of any one of SEQ ID NOs:91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, and 121, or to a fragmentthereof.

In some embodiments, the branched fatty aldehyde, branched fattyalcohol, or derivative thereof is isolated from the host cell, forexample, from the extracellular environment. In certain embodiments, thebranched fatty aldehyde, branched fatty alcohol, or derivative thereofis spontaneously secreted, partially or completely, from the host cell.In alternative embodiments, the branched fatty aldehyde, branched fattyalcohol, or derivative thereof is transported into the extracellularenvironment. In other embodiments, the branched fatty aldehyde, branchedfatty alcohol, or derivative thereof is passively transported into theextracellular environment.

In certain embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell expressing apolynucleotide that hybridizes to a complement of the nucleotidesequence encoding a fatty aldehyde biosynthesis polypeptide listed inTable 6, or to a fragment thereof, wherein the polynucleotide encodes apolypeptide having carboxylic acid reductase activity. In someembodiments, the polypeptide has fatty acid reductase activity.

In some embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell, wherein the geneencoding a fatty acid synthase is modified. In certain embodiments,modifying the expression of a gene encoding a fatty acid synthaseincludes expressing a gene encoding a fatty acid synthase in the hostcell and/or increasing the expression or activity of an endogenous fattyacid synthase in the host cell. In alternate embodiments, modifying theexpression of a gene encoding a fatty acid synthase includes attenuatinga gene encoding a fatty acid synthase in the host cell and/or decreasingthe expression or activity of an endogenous fatty acid synthase in thehost cell. In some embodiments, the fatty acid synthase is athioesterase. In particular embodiments, the thioesterase is encoded bytesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, orfatA1.

In some embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell expressing a geneencoding a fatty alcohol biosynthesis polypeptide. For example, thefatty alcohol biosynthesis polypeptide is an alcohol dehydrogenase. Inparticular embodiments, the fatty alcohol biosynthesis polypeptide isone selected from those listed in Table 8, or a variant thereof.

In any of the above-described embodiments, the microbially producedbranched fatty alcohol and/or derivative thereof is produced by a hostcell, which is genetically engineered to express an attenuated level ofa fatty acid degradation enzyme relative to a wild type host cell. Insome embodiments, the host cell is genetically engineered to express anattenuated level of an acyl-CoA synthase relative to a wild type hostcell. In particular embodiments, the host cell expresses an attenuatedlevel of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL,Pfl-4354, EAV15023, fadD1, fadD2, RPC_4074, fadDD35, fadDD22, faa3p orthe gene encoding the protein ZP_01644857. In certain embodiments, thegenetically engineered host cell comprises a knockout of one or moregenes encoding a fatty acid degradation enzyme, such as theaforementioned acyl-CoA synthase genes. In certain embodiments, the hostcell is genetically engineered to express, relative to a wild type hostcell, a decreased level of at least one of a gene encoding an acyl-CoAdehydrogenase, a gene encoding an outer membrane protein receptor, and agene encoding a transcriptional regulator of fatty acid biosynthesis. Insome embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE.In some embodiments, the gene encoding an outer membrane proteinreceptor is tonA (also known as fhuA). Yet in other embodiments, thegene encoding a transcriptional regulator of fatty acid biosynthesis isfabR.

In yet other embodiments, the microbially produced branched fattyalcohol and/or derivative thereof is produced by a host cell, which isgenetically engineered to express an attenuated level of adehydratase/isomerase enzyme, such as an enzyme encoded by fabA or by agene listed in Table 1 or Table 2. In some embodiments, the host cellcomprises a knockout of fabA or a gene listed in Table 1 or Table 2. Inother embodiments, the host cell is genetically engineered to express anattenuated level of a ketoacyl-ACP synthase, such as an enzyme encodedby fabB or by a gene listed in Table 3 or Table 4. In certainembodiments, the host cell comprises a knockout of fabB or a gene listedin Table 3 or Table 4. In yet other embodiments, the host cell isgenetically engineered to express a modified level of a gene encoding adesaturase enzyme, such as desA.

In some embodiments, the branched fatty alcohol or a derivative thereofis isolated from the host cell, for example, from the extracellularenvironment. In certain embodiments, the branched fatty alcohol or thederivative thereof is spontaneously secreted, partially or completely,from the host cell. In alternative embodiments, the branched fattyalcohol or the derivative thereof is transported into the extracellularenvironment. In other embodiments, the branched fatty alcohol or thederivative thereof is passively transported into the extracellularenvironment.

In some embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell expressing apolynucleotide that hybridizes to a complement of a nucleotide sequenceencoding an acyl-ACP reductases selected from those listed in Table 7,or to a fragment thereof, wherein the polynucleotide encodes apolypeptide having reductase activity. The polynucleotide hybridizesunder low stringency, medium stringency, high stringency, or very highstringency conditions, to a complement of the nucleotide sequenceencoding an acyl-ACP reductases selected from those listed in Table 7,or to a fragment thereof.

In some embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell expressing a geneencoding a fatty alcohol biosynthesis polypeptide in the host cell. Forexample, the fatty alcohol biosynthesis polypeptide is an alcoholdehydrogenase. In particular embodiments, the fatty alcohol biosynthesispolypeptide is one selected from those listed in Table 8, or a variantthereof.

In any of the above-described embodiments, the microbially producedbranched fatty alcohol and/or derivative thereof is produced by a hostcell, which is genetically engineered to express an attenuated level ofa fatty acid degradation enzyme relative to a wild type host cell. Insome embodiments, the host cell is genetically engineered to express anattenuated level of an acyl-CoA synthase relative to a wild type hostcell. In particular embodiments, the host cell expresses an attenuatedlevel of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL,Pfl-4354, EAV15023, fadD1, fadD2, RPC_4074, fadDD35, fadDD22, faa3p orthe gene encoding the protein ZP_01644857. In certain embodiments, thegenetically engineered host cell comprises a knockout of one or moregenes encoding a fatty acid degradation enzyme, such as theaforementioned acyl-CoA synthase genes. In certain embodiments, the hostcell is genetically engineered to express, relative to a wild type hostcell, a decreased level of at least one of a gene encoding an acyl-CoAdehydrogenase, a gene encoding an outer membrane protein receptor, and agene encoding a transcriptional regulator of fatty acid biosynthesis. Insome embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE.In some embodiments, the gene encoding an outer membrane proteinreceptor is tonA (also known as fhuA). Yet in other embodiments, thegene encoding a transcriptional regulator of fatty acid biosynthesis isfabR.

In yet other embodiments, the microbially produced branched fattyalcohol and/or derivative thereof is produced by a host cell, which isgenetically engineered to express an attenuated level of adehydratase/isomerase enzyme, such as an enzyme encoded by fabA or by agene listed in Table 1 or Table 2. In some embodiments, the host cellcomprises a knockout of fabA or a gene listed in Table 1 or Table 2. Inother embodiments, the host cell is genetically engineered to express anattenuated level of a ketoacyl-ACP synthase, such as an enzyme encodedby fabB or by a gene listed in Table 3 or Table 4. In certainembodiments, the host cell comprises a knockout of fabB or a gene listedin Table 3 or Table 4. In yet other embodiments, the host cell isgenetically engineered to express a modified level of a gene encoding adesaturase enzyme, such as desA.

In some embodiments, the branched fatty alcohol or derivative thereof isisolated from the host cell, e.g., from the extracellular environment.In certain embodiments, the branched fatty alcohol or derivative thereofis spontaneously secreted, partially or completely, from the host cell.In alternative embodiments, the branched fatty alcohol or derivativethereof is transported into the extracellular environment. In otherembodiments, the branched fatty alcohol or derivative thereof ispassively transported into the extracellular environment.

In some embodiments, the branched-chain alpha-keto acid dehydrogenasecomplex, the beta ketoacyl-ACP synthase polypeptide, the aldehydebiosynthesis polypeptide, the fatty acid synthase, the acyl-ACPreductase, the alcohol biosynthesis polypeptide, and the fatty aciddegradation enzyme polypeptide are each independently from a bacterium,a plant, an insect, a yeast, a fungus, or a mammal. For example, thebranched-chain alpha-keto acid dehydrogenase complex polypeptides can befrom a bacterium that uses branched amino acids as carbon source,including, for example, Pseudomonas putida, or Bacillus subtilis. Inanother example, the branched-chain alpha-keto acid dehydrogenasecomplex polypeptide can be from a bacterium that comprises branchedfatty acids in its phospholipids, including, for example, a Legionella,Stenotrophomonas, Alteromonas, Flavobacterium, Myxococcus, Bccteroides,Micrococcus, Staphylococcus, Bacillus, Clostridium, Listeria,Lactococcus, or Streptomyces bacterium. In some embodiments, thebacterium is a Leginella pneumophila, Stenotrophomonas maltophilia,Alteromonas macleodii, Flabobacterium phsychrophilum, MyxococcusXanthus, Bacteroides thetaiotaomicron, Macrococcus luteus,Staphylococcus aureus, Clostridium thermocellum, Listeria monocytogenes,Streptomyces lividans, Streptomyces coelicolor, Streptomycesglaucescens, Streptococcus pneumoniae, Streptomyces peucetius,Streptococcus pyogenes, Escherichia coli, Escherichia coli K-12,Lactococcus lactis ssp. Lactis, Mycobacterium tuberculosis, Enterococcustuberculosis, Bacillus subtilis, Lactobacillus plantarum. In someembodiments, suitable fatty aldehyde biosynthesis polypeptides, fattyalcohol biosynthesis polypeptides, acyl-ACP reductases, and otherpolypeptides of the invention can be from a mycobacterium selected fromthe group consisting of Mycobacterium smegmatis, Mycobacteriumabscessus, Mycobacterium avium, Mycobacterium bovis, Mycobacteriumtuberculosis, Mycobacterium leprae, Mycobacterium marinum, andMycobacterium ulcerans. In other embodiments, the bacterium is Nocardiasp. NRRL 5646, Nocardia farcinica, Streptomyces griseus, Salinisporaarenicola, or Clavibacter michiganenesis. In yet further embodiments,the polypeptide of the invention is derived from a cyanobacterium,including, for example, Synechococcus elongatus PCC7942, Synechocystissp. PCC6803, Cyanothece sp. ATCC51142, Prochlorococcus marinus subsp.pastoris str. CCMP1986 PMM0533, Gloeobacter violaceus PCC7421, Nostocpunctiforme PCC73102, Anabaena variabilis ATCC29413, Synechococcuselongatus PCC6301, and Nostoc sp. PCC 7120, Microcoleus chthonoplastesPCC7420, Arthrospira maxima CS-328, Lyngbya sp. PCC8106, Nodulariaspumigena CCY9414, Trichodesmium erythraeum IMS101, Microcystisaeruginosa, Nostoc azollae, Anabaena variabilis, Crocophaera watsonii,Thermosynechococcus elongatus, Gloeobacer violaceus, Cyanobium, orProchlorococcus marinus.

In some embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell cultured in thepresence of at least one biological substrate for the branched-chainalpha-keto acid dehydrogenase polypeptides, the beta ketoacyl-ACPsynthase polypeptide, the aldehyde biosynthesis polypeptide, theacyl-ACP reductase, or the alcohol biosynthesis polypeptide. In someembodiments, the host cell is cultured under conditions that allow theexpression of the branched-chain alpha-keto acid dehydrogenasepolypeptides, the beta ketoacyl-ACP synthase, the aldehyde biosynthesispolypeptide, the acyl-ACP reductase, and/or the alcohol biosynthesispolypeptide. In particular embodiments, the host cell is cultured underconditions that allow the production of branched fatty alcohols orderivatives thereof.

In some embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell cultured in thepresence of at least one biological substrate for the branched-chainalpha-keto acid dehydrogenase complex, the aldehyde biosynthesispolypeptide, the alcohol biosynthesis polypeptide, and/or the acyl-ACPreductase polypeptide. Accordingly, the host cell is cultured underconditions that allow expression of branched-chain alpha-keto aciddehydrogenase complex, the aldehyde biosynthesis polypeptide, thealcohol biosynthesis polypeptide, and/or the acyl-ACP reductasepolypeptide.

In some embodiments, the branched fatty alcohol or derivative thereof isisolated from the host cell, e.g., from the extracellular environment.In some embodiments, the branched fatty alcohol or derivative thereof isspontaneously secreted, partially or completely, from the host cell. Inalternative embodiments, the branched fatty alcohol or derivativethereof is transported into the extracellular environment. In otherembodiments, the branched fatty alcohol or derivative thereof ispassively transported into the extracellular environment.

In another aspect, the invention features a surfactant or detergentcomposition comprising a microbially produced branched fatty alcohol ora derivative thereof. In certain embodiments, the microbially producedbranched fatty alcohol and/or derivative thereof is produced by a hostcell expressing one or more recombinant vectors comprising at least theE1 alpha and beta subunits of a branched-chain alpha-keto aciddehydrogenase. In certain embodiments, the recombinant vector furthercomprises an E2 subunit of a branched-chain alpha-keto aciddehydrogenase. The subunits can be introduced into the host cell inseparate vectors or together in a single vector. For example, the vectorcan comprise a first polynucleotide sequence having at least about 30%,e.g., at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 80%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%identity sequence identity to a polynucleotide sequence listed in FIG.2A, and a second polynucleotide sequence having at least about 30%,e.g., at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 80%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%identity sequence identity to a polynucleotide sequence listed in FIG.2B. In another example, the vector can further comprise a thirdpolynucleotide having at least about 30%, e.g., at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or 100% identity sequence identity to apolynucleotide sequence listed in FIG. 2C. The polynucleotides encodingthe alpha and beta subunits of the E1 subunit can be linked andconstitute a single operon, or they may be separately introduced into avector and/or into a host cell. Likewise, the polynucleotides encodingthe E1 subunit and the polynucleotide encoding the E2 subunit can belinked and constitute a single operon, or they may be separatelyintroduced into a vector and/or into a host cell. For example, a firstvector can comprise a first polynucleotide sequence having at leastabout 30%, e.g., at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 80%, at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or 100%identity sequence identity to a polynucleotide sequence listed in FIG.2A, and a second vector can comprise a second polynucleotide having atleast about 30%, e.g., at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or 100% identity sequence identity to a polynucleotide sequence listedin FIG. 2B.

In some embodiment, the nucleotide sequence of the first polynucleotidehas at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%sequence identity to the nucleotide sequence of any one of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14 and 16; the second polynucleotide has at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99% sequenceidentity to the nucleotide sequence of any one of SEQ ID NOs: 25, 27,29, 31, 33, 35, 37, and 39; and the nucleotide sequence of the thirdpolynucleotide has at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99% sequence identity to the nucleotide sequence of anyone of SEQ ID NOs: 48, 50, 52, 54, 56, 58, 60, and 62. In someembodiment, the nucleotide sequence of the first polynucleotide is anyone of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16, the nucleotidesequence of the second polynucleotide is any one of SEQ ID NOs: 25, 27,29, 31, 33, 35, 37, and 39, and the nucleotide sequence of the thirdpolynucleotide, when present, is any one of SEQ ID NOs: 48, 50, 52, 54,56, 58, 60, and 62.

In some embodiment, each of the vectors above, or another vector cancomprise a fourth polynucleotide sequence having at least about 30%,e.g., at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 80%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%sequence identity to a polynucleotide sequence listed in FIG. 2D. Incertain embodiments, the nucleotide sequence of the fourthpolynucleotide has at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99% sequence identity to the nucleotide sequence of anyone of SEQ ID NOs:70, 72, 74, 76, 78, 80, 82, and 84. In someembodiments, the nucleotide sequence of the fourth polynucleotide is anyone of SEQ ID NOs:70, 72, 74, 76, 78, 80, 82, and 84.

In some embodiments, each of the vectors above, or another vector can beintroduced into the host cell wherein the vector comprises abeta-ketoacyl ACP synthase nucleotide that has at least about 30%, e.g.,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 80%, at least about 85%, at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, at least about 99%, or 100% sequenceidentity to a polynucleotide sequence listed in FIG. 2E. In certainembodiments, the nucleotide sequence of the beta-ketoacyl ACP synthasenucleotide has at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, atleast about 99% sequence identity to the nucleotide sequence of any oneof SEQ ID NOs:91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,115, 117, 119, and 121. In some embodiments, the nucleotide sequence ofbeta-ketoacyl ACP synthase nucleotide is SEQ ID NOs: 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, and 121.

In yet another embodiment, an individual vector comprising abeta-ketoacyl-ACP synthase nucleotide that has at least about 30%, e.g.,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 80%, at least about 85%, at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, at least about 99%, or 100% sequenceidentity to a polynucleotide sequence listed in FIG. 2E can beintroduced into a suitable host cell, independent of whether one or moreother vectors comprising one or more subunits of a branched-chainalpha-keto acid dehydrogenase is introduced into the same cell. Forexample, the host cell can suitably be one that expresses an endogenousbranched-chain alpha-keto acid dehydrogenase, or one or more subunitsthereof.

In some embodiments, the branched fatty aldehyde, branched fattyalcohol, or derivative thereof is isolated from the host cell, forexample, from the extracellular environment. In some embodiments, thebranched fatty aldehyde, branched fatty alcohol or derivative thereof isspontaneously secreted, partially or completely, from the host cell. Inalternative embodiments, the branched fatty aldehyde, branched fattyalcohol or derivative thereof is transported into the extracellularenvironment. In other embodiments, the branched fatty aldehyde, branchedfatty alcohol, or derivative thereof is passively transported into theextracellular environment.

The recombinant vector can further comprises a promoter operably linkedto the nucleotide sequence. In certain embodiments, the promoter is adevelopmentally-regulated, an organelle-specific, a tissue-specific, aninducible, a constitutive, or a cell-specific promoter.

In other embodiments, the recombinant vector comprises at least onesequence selected from the group consisting of (a) a regulatory sequenceoperatively coupled to the nucleotide sequence; (b) a selection markeroperatively coupled to the nucleotide sequence; (c) a marker sequenceoperatively coupled to the nucleotide sequence; (d) a purificationmoiety operatively coupled to the nucleotide sequence; (e) a secretionsequence operatively coupled to the nucleotide sequence; and (f) atargeting sequence operatively coupled to the nucleotide sequence.

In some embodiments, the recombinant vector is a plasmid.

In some embodiments, the host cell expresses a polypeptide encoded bythe recombinant vector. In some embodiments, the nucleotide sequence isstably incorporated into the genomic DNA of the host cell, and theexpression of the nucleotide sequence is under the control of aregulated promoter region. In an exemplary embodiment, one or more ofthe polynucleotides encoding a branched-chain alpha-keto aciddehydrogenase polypeptide, a beta ketoacyl-ACP synthase polypeptide, afatty aldehyde biosynthesis polypeptide, a fatty alcohol biosynthesispolypeptide, and/or an acyl-ACP reductase of the invention can be stablyincorporated into the genomic DNA of the host cell, and the expressionof the polynucleotide sequence is under the control of a regulatedpromoter region.

In some embodiment, an above-described vector or another vector can beintroduced into the host cell wherein the vector comprises a fattyaldehyde biosynthesis polynucleotide having at least about 70% sequenceidentity to a nucleotide sequence encoding an enzyme listed in Table 6.

In some embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell wherein theexpression of a gene encoding a fatty acid synthase is modified. Incertain embodiments, modifying the expression of a gene encoding a fattyacid synthase includes expressing a gene encoding a fatty acid synthasein the host cell and/or increasing the expression or activity of anendogenous fatty acid synthase in the host cell. In alternateembodiments, modifying the expression of a gene encoding a fatty acidsynthase includes attenuating a gene encoding a fatty acid synthase inthe host cell and/or decreasing the expression or activity of anendogenous fatty acid synthase in the host cell. In some embodiments,the fatty acid synthase is a thioesterase. In particular embodiments,the thioesterase is encoded by tesA, tesA without leader sequence, tesB,fatB, fatB2, fatB3, fatA, or fatA1.

In some embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell expressing a geneencoding a fatty alcohol biosynthesis polypeptide. For example, thefatty alcohol biosynthesis polypeptide is an alcohol dehydrogenase. Inparticular embodiments, the fatty alcohol biosynthesis polypeptidecomprises the amino acid sequence of an enzyme listed in Table 8, or avariant thereof.

In any of the embodiments described above, the microbially producedbranched fatty alcohol and/or derivative thereof is produced by a hostcell, which is genetically engineered to express an attenuated level ofa fatty acid degradation enzyme relative to a wild type host cell. Insome embodiments, the host cell is genetically engineered to express anattenuated level of an acyl-CoA synthase relative to a wild type hostcell. In particular embodiments, the host cell expresses an attenuatedlevel of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL,Pfl-4354, EAV15023, fadD1, fadD2, RPC_4074, fadDD35, fadDD22, faa3p orthe gene encoding the protein ZP_01644857. In certain embodiments, thegenetically engineered host cell comprises a knockout of one or moregenes encoding a fatty acid degradation enzyme, such as theaforementioned acyl-CoA synthase genes. In certain embodiments, the hostcell is genetically engineered to express, relative to a wild type hostcell, a decreased level of at least one of a gene encoding an acyl-CoAdehydrogenase, a gene encoding an outer membrane protein receptor, and agene encoding a transcriptional regulator of fatty acid biosynthesis. Insome embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE.In some embodiments, the gene encoding an outer membrane proteinreceptor is tonA (also known as fhuA). Yet in other embodiments, thegene encoding a transcriptional regulator of fatty acid biosynthesis isfabR.

In yet other embodiments, the microbially produced branched fattyalcohol and/or derivative thereof is produced by a host cell, which isgenetically engineered to express an attenuated level of adehydratase/isomerase enzyme, such as an enzyme encoded by fabA or by agene listed in Table 1 or Table 2. In some embodiments, the host cellcomprises a knockout of fabA or a gene listed in Table 1 or Table 2. Inother embodiments, the host cell is genetically engineered to express anattenuated level of a ketoacyl-ACP synthase, such as an enzyme encodedby fabB or by a gene listed in Table 3 or Table 4. In certainembodiments, the host cell comprises a knockout of fabB or a gene listedin Table 3 or Table 4. In yet other embodiments, the host cell isgenetically engineered to express a modified level of a gene encoding adesaturase enzyme, such as desA.

In certain other embodiments, any of the vectors comprising the E1alpha, E1 beta, and/or optionally E2 and/or optionally E3 subunits of abranched-chain alpha-keto acid dehydrogenase complex or another vectorcan be introduced into the host cell wherein the vector furthercomprises an acyl-ACP reductase polynucleotide having at least about 70%sequence identity to a nucleotide sequence encoding an enzyme listed inTable 7.

In some embodiments, the host cell is cultured in the presence of atleast one biological substrate for the branched-chain alpha-keto aciddehydrogenase complex, the aldehyde biosynthesis polypeptide, thealcohol biosynthesis polypeptide, and/or the acyl-ACP reductasepolypeptide. In certain embodiments, the host cell is cultured underconditions that are sufficient for expressing a branched-chainalpha-keto acid dehydrogenase complex, an aldehyde biosynthesispolypeptide, an alcohol biosynthesis polypeptide, and/or an acyl-ACPreductase polypeptide. In certain other embodiments, the host cell iscultured under conditions that allow the production of branched fattyalcohols or derivatives thereof.

In some embodiments, the microbially produced branched fatty alcoholand/or derivative thereof is produced by a host cell cultured in thepresence of at least one biological substrate for the branched-chainalpha-keto acid dehydrogenase complex, the aldehyde biosynthesispolypeptide, the alcohol biosynthesis polypeptide, and/or the acyl-ACPreductase polypeptide. Accordingly, the host cell is cultured underconditions that allow expression of branched-chain alpha-keto aciddehydrogenase complex, the aldehyde biosynthesis polypeptide, thealcohol biosynthesis polypeptide, and/or the acyl-ACP reductasepolypeptide.

In some embodiments, the branched fatty alcohol or derivative thereof isisolated from the host cell, for example, from the extracellularenvironment. In some embodiments, the branched fatty alcohol orderivative thereof is secreted from the host cell. In alternativeembodiments, the branched fatty alcohol or derivative thereof istransported into the extracellular environment. In other embodiments,the branched fatty alcohol or derivative thereof is passivelytransported into the extracellular environment.

In any of the aspects of the invention described herein, the host cellcan be selected from the group consisting of a mammalian cell, plantcell, insect cell, yeast cell, fungus cell, filamentous fungi cell, andbacterial cell. In some embodiments, the host cell is a Gram-positivebacterial cell. In other embodiments, the host cell is a Gram-negativebacterial cell. In some embodiments, the host cell is selected from thegenus Escherichia, Bacillus, Lactobacillus, Rhodococcus, Pseudomonas,Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor,Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete,Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas,Schizosaccharomyces, Yarrowia, or Streptomyces.

In certain embodiments, the host cell is a Bacillus lentus cell, aBacillus brevis cell, a Bacillus stearothermophilus cell, a Bacilluslicheniformis cell, a Bacillus alkalophilus cell, a Bacillus coagulanscell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillusthuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell,a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell.

In other embodiments, the host cell is a Trichoderma koningii cell, aTrichoderma viride cell, a Trichoderma reesei cell, a Trichodermalongibrachiatum cell, an Aspergillus awamori cell, an Aspergillusfumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulanscell, an Aspergillus niger cell, an Aspergillus oryzae cell, a Humicolainsolens cell, a Humicola lanuginose cell, a Rhodococcus opacus cell, aRhizomucor miehei cell, or a Mucor michei cell.

In yet other embodiments, the host cell is a Streptomyces lividans cellor a Streptomyces murinus cell.

In yet other embodiments, the host cell is an Actinomycetes cell.

In some embodiments, the host cell is a Saccharomyces cerevisiae cell.

In particular embodiments, the host cell is a cell from an eukaryoticplant, algae, cyanolacterium, green-sulfur bacterium, green non-sulfurbacterium, purple sulfur bacterium, purple non-sulfur bacterium,extremophile, yeast, fungus, engineered organisms thereof, or asynthetic organism. In some embodiments, the host cell is lightdependent or fixes carbon. In some embodiments, the host cell is lightdependent or fixes carbon. In some embodiments, the host cell hasautotrophic activity. In some embodiments, the host cell hasphotoautotrophic activity, such as in the presence of light. In someembodiments, the host cell is heterotrophic or mixotrophic in theabsence of light. In certain embodiments, the host cell is a cell fromAvabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, Zea mays,Botryococcuse braunii, Chlamydomonas reinhardtii, Dunaliela salina,Synechococcus Sp. PCC 7002, Synechococcus Sp. PCC 7942, SynechocystisSp. PCC 6803, Thermosynechococcus elongates BP-1, Chlorobium tepidum,Chloroflexus auranticus, Chromatiumm vinosum, Rhodospirillum rubrum,Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridiumljungdahlii, Clostridiuthermocellum, Penicillium chrysogenum, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pseudomonas fluorescens, or Zymomonas mobilis.

In other embodiments, the host cell is a CHO cell, a COS cell, a VEROcell, a BHK cell, a HeLa cell, a Cv1 cell, an MDCK cell, a 293 cell, a3T3 cell, or a PC12 cell.

In yet other embodiments, the host cell is an E. coli cell. In certainembodiments, the E. coli cell is a strain B, a strain C, a strain K, ora strain W E. coli cell.

In further embodiments, the host cell can be genetically engineered toexpress an attenuated level of a dehydratase/isomerase enzyme. Forexample, an E. coli cell is chosen as a suitable host cell, wherein oneor more of the endogenous dehydratase/isomerase enzymes such as thoselisted in Table 1 below can be attenuated or knocked out.

TABLE 1 E.coli dehydratase/isomerase enzymes Polynucleotide PolypeptideGene Name Acc. No. Acc. No. fabA beta-hydroxydecanoyl GU072596.1ACY27485.1 thioester dehydrase fabZ (3R)-hydroxymyristol acyl GU072604ACY27493.1 carrier protein dehydratase cysM cysteine synthase BCP001637.1 ACX38914 maoC fused aldehyde dehydroge- CP001637 ACX39905.1nase/enoyl-CoA hydratase

Other dehydratase/isomerase enzymes encoded by a gene listed below inTable 2 can also be attenuated or knocked from an organism comprisingsuch a gene.

TABLE 2 Other dehydatase/isomerase enzymes Organism Accession No.Shigella sp. D9 ZP_05432652 Citrobacter youngae ATCC 29220 ZP_04561391.1Salmonella enterica YP_001570967.1 Escherichia fergusonii ATCC 35469YP_002382254.1 Klebsiella pneumoniae NTUH-K2044 YP_002918743.1Enterobacter cancerogenus ATCC 35316 ZP_03281954.1 Cronobacterturicensis CBA29728.1 Erwinia pyrifoliae Ep1/96 YP_002649242.1Pectobacterium carotovorum YP_003018119.1 subsp. carotovorum PC1 Dickeyadadantii Ech703 YP_002987184.1 Edwardsiella ictaluri 93-146YP_002932813.1 Providencia alcalifaciens DSM 30120 ZP_03317956.1Yersinia kristensenii ATCC 33638 ZP_04624337.1 Photorhabdus asymbioticaYP_003041580.1 Pantoea sp. At-9b ZP_05728924.1 Actinobacillussuccinogenes 130Z YP_001344737.1 Mannheimia succiniciproducens MBEL55EYP_088386.1 Pasteurella multocida subsp. NP_245421.1 multocida str. Pm70Haemophilus somnus 129PT YP_719117.1 Proteus mirabilis HI4320YP_002150544.1 Sodalis glossinidius str. ‘morsitans’ YP_454706.1Candidatus Blochmannia YP_277927.1 pennsylvanicus str. BPENAggregatibacter aphrophilus NJ8700 YP_003007342.1 Vibrio cholerae MZO-3ZP_01958381.1 Baumannia cicadellinicola str. Hc YP_588853.1 (Homalodiscacoagulata) Vibrionales bacterium SWAT-3 ZP_01815187.1 Aliivibriosalmonicida LFI1238 YP_002262988.1 Aeromonas salmonicida subsp.YP_001141819.1 salmonicida A449 Wigglesworthia glossinidia endosymbiontNP_871303.1 of Glossina brevipalpis Glaciecola sp. HTCC2999ZP_03560821.1 Alteromonas macleodii ATCC 27126 ZP_04714556.1

In other embodiments, the host cell is genetically engineered to expressan attenuated level of an endogenous ketoacyl-ACP synthase. For example,an E. coli cell is used as a suitable host cell, wherein one or more ofthe ketoacyl-ACP genes listed in Table 3 below can be attenuated orknocked out.

TABLE 3 E.coli ketoacyl-ACP synthase enzymes Polynucleotide PolypeptideGene Name Acc. No. Acc. No. fabB B-ketoacyl synthase/ GU072597.1ACY27486.1 3-oxoacyl-[acyl-carrier-pro tein] synthase I fabF3-oxoacyl-[acyl-carrier- GU072598.1 ACY27487 protein] synthase II fadJfused enoyl-CoA hydratase CP001637.1 ACX38989.1 and epimerase/isomerase/3-hydroxyacyl-CoA dehydrogenase xerC site-specific tyrosine CP001637.1ACX41768.1 recombinase yqeF predicted acyltransferase CP001637.1ACX38529.1 murQ predicted PTS component CP001637.1 ACX38907.1

Other endogenous ketoacyl-ACP synthases, such as the ones listed inTable 4, can be attenuated or knocked out from an organism comprisingsuch an enzyme.

TABLE 4 Other ketoacyl-ACP synthases Organism Accession No. Shigellaboydii CDC 3083-94 YP_001881145.1 Escherichia fergusonii ATCC 35469YP_002382013.1 Salmonella enterica subsp. arizonae YP_001569590.1Citrobacter sp. 30_2 ZP_04562837.1 Klebsiella pneumoniae subsp.pneumoniae YP_001336360.1 MGH 78578 Pectobacterium carotovorum subsp.ZP_03831287.1 carotovorum WPP14 Enterobacter cancerogenus ATCC 35316ZP_03283474.1 Pantoea sp. At-9b ZP_05730617.1 Cronobacter turicensisCBA32510.1 Dickeya dadantii Ech586 ZP_05723897.1 Erwinia tasmaniensisEt1/99 YP_001907100.1 Serratia proteamaculans 568 YP_001479594.1Edwardsiella ictaluri 93-146 YP_002934130.1 Sodalis glossinidius str.‘morsitans’ YP_455303.1 Yersinia aldovae ATCC 35236 ZP_04620215.1Providencia stuartii ATCC 25827 ZP_02961167.1 Photorhabdus asymbioticaYP_003040275.1 Proteus mirabilis HI4320 YP_002151524.1 CandidatusBlochmannia pennsylvanicus str. BPEN YP_278005.1 Glaciecola sp. HTCC2999ZP_03561088.1 Vibrio cholerae V51 ZP_04919940.1 Wigglesworthiaglossinidia endosymbiont of NP_871411.1 Glossina brevipalpis Tolumonasauensis DSM 9187 YP_002892770.1 Actinobacillus pleuropneumoniae serovar1 str. 4074 ZP_00134992.2 Aggregatibacter aphrophilus NJ8700YP_003007711.1 Pseudoalteromonas tunicata D2 ZP_01135065.1 Vibrionalesbacterium SWAT-3 ZP_01816638.1 Pasteurella multocida subsp. multocidastr. Pm70 NP_245276.1 Mannheimia succiniciproducens MBEL55E YP_088783.1Haemophilus somnus 129PT YP_718877.1 Shewanella loihica PV-4YP_001094535.1 Aliivibrio salmonicida LFI1238 YP_002262558.1

In yet other embodiments, the host cell is genetically engineered toexpress a modified level of a gene encoding a desaturase enzyme, such asdesA.

In certain embodiments, the microorganism is genetically engineered toexpress a modified level (including, e.g., to attenuate or knock out orto express or overexpress) of a gene encoding a fatty aldehydebiosynthesis polypeptide. In some embodiments, the fatty aldehydebiosynthesis polypeptide comprises an amino acid sequence that has atleast 70% sequence identity to an enzyme listed in Table 6.

In certain embodiments, the microorganism is genetically engineered toexpress a modified level of a fatty acid synthase in the host cell. Anexemplary fatty acid synthase is a thioesterase encoded by, for example,tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, orfatA1.

In certain embodiments, the microorganism is genetically engineered toexpress a modified level of gene encoding a fatty alcohol biosynthesispolypeptide. For example, the fatty alcohol biosynthesis polypeptide isan alcohol dehydrogenase. In particular embodiments, the fatty alcoholbiosynthesis polypeptide comprises an amino acid sequence that has atleast 70% sequence identity to an enzyme listed in Table 8.

Branched-Chain Alpha-Keto Acid Dehydrogenase Complex (BKD Complex) andBeta Ketoacyl-ACP Synthase

The methods described herein can be used to produce branched fattyalcohols and/or derivatives, for example, from alpha keto acids. Theoxidative decarboxylation step, which converts the alpha keto acids tothe corresponding branched-chain acyl-CoA involves a branched-chainα-keto acid dehydrogenase complex (bkd; EC 1.2.4.4.) (Denoya et al., J.Bacteriol. 177:3504 (1995)), which consists of E1 alpha/beta(decarboxylase), E2 (dihydrolipoyl transacylase), and E3 (dihydrolipoyldehydrogenase) subunits. Any microorganism that possesses branched-chainfatty acids, and/or grows on branched-chain amino acids can be used as asource to isolate bkd genes for expression in host cells, for example,E. coli. Furthermore, E. coli has the E3 component as part of itspyruvate dehydrogenase complex (lpd, EC 1.8.1.4, GenBank accessionNP_414658). Thus, branched fatty alcohols and/or derivatives can be madeby heterologously expressing only the E1 alpha/beta and E2 bkd genes.Furthermore, certain of the host cells, including E. coli, can producebranched products when only the E1 alpha/beta is expressed withoutco-expression of the E2 bkd gene.

On the other hand, microorganisms that endogenously express a suitablebeta-ketoacyl ACP synthase can be engineered to express or overexpressat least the first (E1) subunit of a branched-chain alpha keto aciddehydrogenase complex, optionally also the second (E2) and/or the third(E3) subunits of that complex to produce the desirable branched fattyalcohols and/or derivatives thereof. The endogenous beta-ketoacyl ACPsynthase can be overexpressed, or can be modified such that it isattenuated or deleted, and a heterologous beta-ketoacyl ACP synthasegene can be expressed in its place.

In a further embodiment, microorganisms that endogenously express atleast the first (E1) subunit of a branched-chain alpha keto aciddehydrogenase complex, and optionally also the second (E2) and/or thethird (E3) subunits of that complex, can be engineered to express oroverexpress a beta-ketoacyl ACP synthase. For example, the endogenousgenes encoding the subunits of the branched-chain alpha keto aciddehydrogenase complex can be overexpressed, or can be modified such thatthey are attenuated or deleted and a gene encoding one or more subunitsof a heterologous branched-chain alpha keto acid dehydrogenase complexcan be expressed in the host cell.

Substrates for Branched Fatty Alcohol Production

The branched fatty alcohols and/or derivatives, as well as thesurfactant compositions comprising them, can be produced from, forexample, branched fatty aldehydes, which themselves can be produced froman appropriate substrate. While not wishing to be bound by theory, it isbelieved that the branched fatty aldehyde biosynthetic polypeptidesdescribed herein produce branched fatty aldehydes from substrates via areduction mechanism. In some instances, the substrate is a branchedfatty acid derivative, and a fatty aldehyde having particular branchingpatterns and carbon chain length can be produced from a branched fattyacid derivative having those characteristics. The branched fattyaldehyde can then be converted into the desired branched fatty alcoholin a reaction catalyzed by a fatty alcohol biosynthesis polypeptide.

Alternatively, a suitable acyl-ACP reductases can be employed to converta branched acyl-ACP into a fatty aldehyde, which can in turn beconverted into a branched fatty alcohol in a reaction catalyzed by afatty alcohol biosynthesis polypeptide.

Accordingly, each step within a biosynthetic pathway that leads to theproduction of a branched fatty acid derivative substrate can be modifiedto produce or overproduce the branched substrate of interest. Forexample, known genes involved in the fatty acid biosynthetic pathway orthe fatty aldehyde biosynthesis pathway can be expressed, overexpressed,or attenuated in host cells to produce a desired substrate (see, e.g.,International Publication WO 2008/119082, the disclosure of which isincorporated by reference).

Synthesis of Branched Fatty Alcohols and Substrates

Fatty acid synthase (FAS) is a group of polypeptides that catalyze theinitiation and elongation of acyl chains (Marrakchi et al., BiochemicalSociety, 30: 1050-1055 (2002)). The acyl carrier protein (ACP) alongwith the enzymes in the FAS pathway control the length, degree ofsaturation, and branching of the fatty acid derivatives produced. Thefatty acid biosynthetic pathway involves the precursors acetyl-CoA andmalonyl-CoA. The steps in this pathway are catalyzed by enzymes of thefatty acid biosynthesis (fab) and acetyl-CoA carboxylase (acc) genefamilies (see, e.g., Heath et al., Prog. Lipid Res., 40(6): 467-97(2001)).

Host cells can be engineered to express fatty acid derivative substratesby recombinantly expressing or overexpressing one or more fatty acidsynthase genes, such as acetyl-CoA and/or malonyl-CoA synthase genes.For example, to increase acetyl-CoA production, one or more of thefollowing genes can be expressed in a host cell: pdh (a multienzymecomplex comprising aceEF (which encodes the E1p dehydrogenase component,the E2p dihydrolipoamide acyltransferase component of the pyruvate and2-oxoglutarate dehydrogenase complexes, and lpd), panK, fabH, fabB,fabD, fabG, acpP, and fabF. Exemplary GenBank accession numbers forthese genes are: pdh (BAB34380, AAC73227, AAC73226), panK (also known asCoA, AAC76952), aceEF (AAC73227, AAC73226), fabH (AAC74175), fabB(P0A953), fabD (AAC74176), fabG (AAC74177), acpP (AAC74178), and fabF(AAC74179). Additionally, the expression levels of fadE, gpsA, ldhA,pflb, adhE, pta, poxB, ackA, and/or ackB can be attenuated orknocked-out in an engineered host cell by transformation withconditionally replicative or non-replicative plasmids containing null ordeletion mutations of the corresponding genes or by substitutingpromoter or enhancer sequences. Exemplary GenBank accession numbers forthese genes are: fadE (AAC73325), gspA (AAC76632), ldhA (AAC74462), pflb(AAC73989), adhE (AAC74323), pta (AAC75357), poxB (AAC73958), ackA(AAC75356), and ackB (BAB81430). The resulting host cells will haveincreased acetyl-CoA production levels when grown in an appropriateenvironment.

Malonyl-CoA overexpression can be affected by introducing accABCD (e.g.,accession number AAC73296, EC 6.4.1.2) into a host cell. Fatty acidproduction can be further increased by introducing into the host cell aDNA sequence encoding a lipase (e.g., accession numbers CAA89087,CAA98876).

In addition, inhibiting PlsB can lead to an increase in the levels oflong chain acyl-ACP, which will inhibit early steps in the pathway(e.g., accABCD, fabH, and fabI). The plsB (e.g., accession numberAAC77011) D311E mutation can be used to increase the amount of availablefatty acids.

In addition, a host cell can be engineered to overexpress a sfa gene(suppressor of fabA, e.g., accession number AAN79592) to increaseproduction of monounsaturated fatty acids (Rock et al., J. Bacteriology,178: 5382-5387 (1996)).

The chain length of a fatty acid derivative substrate can be selectedfor by modifying the expression of selected thioesterases. Thioesteraseinfluences the chain length of fatty acids produced. Hence, host cellscan be engineered to express, overexpress, have attenuated expression,or not to express one or more selected thioesterases to increase theproduction of a preferred fatty acid derivative substrate. For example,C₁₀ fatty acids can be produced by expressing a thioesterase that has apreference for producing C₁₀ fatty acids and attenuating thioesterasesthat have a preference for producing fatty acids other than C₁₀ fattyacids (e.g., a thioesterase which prefers to produce C₁₄ fatty acids).This would result in a relatively homogeneous population of fatty acidsthat have a carbon chain length of 10. In other instances, C₁₄ fattyacids can be produced by attenuating endogenous thioesterases thatproduce non-C₁₄ fatty acids and expressing the thioesterases that have apreference for C₁₄-ACP. In some situations, C₁₂ fatty acids can beproduced by expressing thioesterases that have a preference for C₁₂-ACPand attenuating thioesterases that preferentially produce non-C₁₂ fattyacids. Acetyl-CoA, malonyl-CoA, and fatty acid overproduction can beverified using methods known in the art, for example, by usingradioactive precursors, HPLC, or GC-MS subsequent to cell lysis.Non-limiting examples of thioesterases that can be used in the methodsdescribed herein are listed in Table 5.

TABLE 5 Thioesterases Accession Number Source Organism Gene AAC73596 E.coli tesA without leader sequence AAC73555 E. coli tesB Q41635, AAA34215Umbellularia california fatB AAC49269 Cuphea hookeriana fatB2 Q39513;AAC72881 Cuphea hookeriana fatB3 Q39473, AAC49151 Cinnamonum camphorumfatB CAA85388 Arabidopsis thaliana fatB [M141T]* NP 189147; NP 193041Arabidopsis thaliana fatA CAC39106 Bradyrhiizobium japonicum fatAAAC72883 Cuphea hookeriana fatA AAL79361 Helianthus annus fatA1 *Mayeret al., BMC Plant Biology, 7: 1-11 (2007)

In certain embodiments, a host cell, which is used to produce branchedfatty alcohols and/or derivatives herein, can be engineered to expressor overexpress one of more fatty aldehyde biosynthetic polypeptides.Alternatively, the host cell can be engineered to express an attenuatedlevel of an endogenous fatty aldehyde biosynthetic polypeptide. In otherinstances, a fatty aldehyde biosynthetic polypeptide, a variant, or afragment thereof is expressed in a host cell that contains a naturallyoccurring mutation that results in an increased level of branched fattyaldehyde substrate in the host cell or of branched fatty alcoholproduced by the host cell. In some instances, a branched fatty aldehydeis produced by expressing a fatty aldehyde biosynthesis gene, forexample, a carboxylic acid reductases gene, encoding a protein listed inTable 6, below, as well as a polynucleotide variant there. In someinstances, the fatty aldehyde biosynthesis gene encodes one of theenzymes listed in Table 6 below.

TABLE 6 Fatty Aldehyde Biosynthesis Genes Name/Organism Accession No.Nocardia sp. NRRL 5646 >gi|40796035|gb|AAR91681.1| Mycobacteriumtuberculosis >gi|15609727|ref|NP_217106.1 H37Rv Mycobacteriumsmegmatis >gi|118174788|gb|ABK75684.1| str. MC2 155 Mycobacteriumsmegmatis >gi|118469671|ref|YP_889972.1| str. MC2 155 FadD9uniprot|A0PPD8|A0PPD8_MYCUA Tsukamurellapaurometabola >gi|22798060|ref|ZP_04027864.1| DSM 20162 Cyanobium sp.PCC 7001 >gi|254431429|ref|ZP_05045132.1| Putative acyl-CoAdehydrogenase >uniprot|A0QIB5|A0QIB5_MYCA1 NADdependent >uniprot|A0QWI7|A0QWI7_MYCS2 epimerase/dehydrataseMycobacterium intracellulare >gi|254819907|ref|ZP_05224908.1| ATCC13950Putative long-chain >uniprot|A0R484|A0R484_MYCS2 fattyacid-CoA ligaseMycobacterium kansasii >gi|240173202|ref|ZP_04751860.1| ATCC 12478Probable fatty-acid-CoA >uniprot|A1KLT8|A1KLT8_MYCBP ligase fadD9Mycobacterium intracellulare >gi|254822803|ref|ZP_05227804.1| ATCC13950Fatty-acid-CoA ligase fadD9 >uniprot|A1QUM2|A1QUM2_MYCTF Thioesterreductase domain >uniprot|A1T887|A1T887_MYCVP Thioester reductasedomain >uniprot|A1UFA8|A1UFA8_MYCSK Mycobacteriumavium >gi|254775919|ref|ZP_05217435.1| subsp. ATCC 25291 Thioesterreductase domain >uniprot|A3PYW9|A3PYW9_MYCSJ Mycobacterium lepraeBr4923 >gi|219932734|emb|CAR70557.1| Putative acyl-CoAsynthetase >uniprot|A5CM59|A5CM59_CLAM3 Thioester reductasedomain >uniprot|A8M8D3|A8M8D3_SALAI Probablefatty-acid-CoA >uniprot|B1MCR9|B1MCR9_MYCAB ligase FadD Probablefatty-acid-CoA >uniprot|B1MCS0|B1MCS0_MYCAB ligase FadD Putativefatty-acid-CoA ligase >uniprot|B1MDX4|B1MDX4_MYCAB Probablefatty-acid-coa >uniprot|B1MLD7|B1MLD7_MYCAB ligase FadD Putativecarboxylic acid reductase >uniprot|B1VMZ4|B1VMZ4_STRGG Fatty-acid-CoAligase FadD9_1 >uniprot|B2HE95|B2HE95_MYCMM Fatty-acid-CoA ligaseFadD9 >uniprot|B2HN69|B2HN69_MYCMM Putative Acyl-CoAsynthetase >uniprot|O69484|O69484_MYCLE Probable peptide synthetasenrp >uniprot|Q10896|Q10896_MYCTU Putative carboxylic acidreductase >uniprot|Q5YY80|Q5YY80_NOCFAATP/NADPH-dependent >uniprot|Q6RKB1|Q6RKB1_9NOCA carboxylic acidreductase FadD9 >uniprot|Q741P9|Q741P9_MYCPA Substrate--CoA ligase,putative >uniprot|Q7D6X4|Q7D6X4_MYCTU Probablefatty-acid-coa >uniprot|Q7TY99|Q7TY99_MYCBO ligase fadd9 Putativeacyl-CoA synthetase >uniprot|Q9CCT4|Q9CCT4_MYCLE Putativeuncharacterized protein >uniprot|Q54JK0|Q54JK0_DICDI Putativenon-ribosomal >uniprot|Q2MFQ3|Q2MFQ3_STRRY peptide synthetaseMycobacterium tuberculosis >gi|215431545|ref|ZP_03429464.1| EAS054Mycobacterium tuberculosis >gi|218754327|ref|ZP_03533123.1| GM 1503Mycobacterium tuberculosis T85 >gi|215446840|ref|ZP_03433592.1|Mycobacterium tuberculosis T17 >gi|219558593|ref|ZP_03537669.1|Mycobacterium intracellulare >gi|254819907|ref|ZP_05224908.1| ATCC13950

In certain embodiments, a host cell, which is used to produce branchedfatty alcohols and/or derivatives herein, can be engineered to expressor overexpress one or more acyl-ACP reductases polypeptides, variants,or fragments thereof to achieve an improved production of one or moredesirable branched fatty alcohols or derivatives. Alternatively, a hostcell can be engineered to express an attenuated level of an endogenousacyl-ACP reductase. Non-limiting examples of suitable acyl-ACPreductases are listed in Table 7 below:

TABLE 7 Acyl-ACP Reductase Polypeptides Organism Accession No.Synechococcus elongatus Synpcc7942_1594 (YP_400611) PCC7942Synechocystis sp. sll0209 (NP_442146) Cyanothece sp. ATCC51142 cce_1430(YP_001802846) Prochlorococcus marinus CCMP1986 PMM0533 (NP_892651)subsp.pastoris str. Gloeobacter violaceus PCC7421 NP_96091 (gll3145)Nostoc punctiforme PCC73102 ZP_00108837 (Npun02004176) Anabaenavariabilis ATCC29413 YP_323044 (Ava_2534) Synechococcus elongatusPCC6301 YP_170761 (syc0051_d) Nostoc sp. PCC 7120 alr5284 (NP_489324)Prochlorococcus marinus CCMP1986 PMM0533 (NP_892651) subsp.pastoris str.

In certain embodiments, a host cell, which is used to produce fattyalcohols and/or derivatives herein, can be further engineered to expressor overexpress one or more fatty alcohol biosynthesis polypeptides,variants, or fragments thereof in order to achieve an improvedproduction of one or more desirable branched fatty alcohols orderivatives. Alternatively, a host cell can be engineered to express anattenuated level of an endogenous fatty alcohol biosynthesispolypeptide. Non limiting examples of suitable fatty alcoholbiosynthesis polypeptides are listed in Table 8 below:

TABLE 8 Fatty Alcohol Biosynthesis/Alcohol Dehydrogenase PolypeptideGenBank GenBank GenBank Name Accession No. Name Accession No. NameAccession No. ygjB NP_418690 YggP YP_026187 YciK NP_415787 yahKNP_414859 YiaY YP_026233 YgfF NP_417378 adhP NP_415995 FucO NP_417279YghA NP_417476 ydjL NP_416290 EutG NP_416948 YjgI NP_418670 ydjJNP_416288 YqhD NP_417484 YdfG NP_416057 idnD NP_418688 AdhE NP_415757YgcW NP_417254 Tdh NP_418073 dkgB NP_414743 UcpA NP_416921 yjjNNP_418778 YdjG NP_416285 EntA NP_415128 rspB NP_416097 YeaE NP_416295FolM NP_416123 gatD NP_416594 dkgA NP_417485 HdhA NP_416136 yphCNP_417040 YajO NP_414953 HcaB NP_417036 yhdH NP_417719 YghZ NP_417474SrlD NP_417185 ycjQ NP_415829 Tas NP_417311 KduD NP_417319 yncBNP_415966 YdhF YP_025305 IdnO NP_418687 Qor NP_418475 YdbC NP_415924FabG NP_415611 frmA NP_414890 ybbO NP_415026 FabI NP_415804 ybdRNP_415141 yohF NP_416641 YdjA NP_416279

In some instances, a host cell, which can be used to produce branchedfatty alcohols and/or derivatives herein, is genetically engineered toincrease the level of branched fatty acids in the host cell relative toa corresponding wild-type host cell. For example, the host cell can begenetically engineered to express a reduced level of an acyl-CoAsynthase relative to a wild-type host cell. In one embodiment, the levelof expression of one or more genes (e.g., an acyl-CoA synthase gene) isreduced by genetically engineering a “knock out” host cell.

Any known acyl-CoA synthase gene can be reduced or knocked out in a hostcell. Non-limiting examples of acyl-CoA synthase genes include fadD,fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_4074, fadDD35,fadDD22, faa3p or the gene encoding the protein ZP_01644857. Specificexamples of acyl-CoA synthase genes include fadDD35 from M. tuberculosisH37Rv [NP_217021], fadDD22 from M. tuberculosis H37Rv [NP_217464], fadDfrom E. coli [NP_416319], fadK from E. coli [YP_416216], fadD fromAcinetobacter sp. ADP1 [YP_045024], fadD from Haemophilus influenzaRdkW20 [NP_438551], fadD from Rhodopseudomonas palustris Bis B18[YP_533919], BH3101 from Bacillus halodurans C-125 [NP_243969], Pfl-4354from Pseudomonas fluorescens Pfo-1 [YP_350082], EAV15023 from Comamonastestosterone KF-1 [ZP_01520072], yhfL from B. subtilis [NP_388908],fadD1 from P. aeruginosa PAO1 [NP_251989], fadD1 from Ralstoniasolanacearum GM1 1000 [NP_520978], fadD2 from P. aeruginosa PAO1[NP_251990], the gene encoding the protein ZP_01644857 fromStenotrophomonas maltophilia R551-3, faa3p from Saccharomyces cerevisiae[NP_012257], faa1p from Saccharomyces cerevisiae [NP_014962], lcfA fromBacillus subtilis [CAA99571], or those described in Shockey et al.,Plant. Physiol., 129: 1710-1722 (2002); Caviglia et al., J. Biol. Chem.,279: 1163-1169 (2004); Knoll et al., J. Biol. Chem., 269(23): 16348-56(1994); Johnson et al., J. Biol. Chem., 269: 18037-18046 (1994); andBlack et al., J. Biol. Chem. 267: 25513-25520 (1992).

Production of Branched Precursors

Branched fatty alcohols and derivatives can be produced from branchedfatty aldehydes containing one or more branched points, using branchedacyl-ACPs as substrates for a fatty aldehyde biosynthesis polypeptide oran acyl-ACP reductase polypeptide as described herein. The first step informing branched fatty alcohol precursors is the production of thecorresponding alpha-keto acids by a branched-chain amino acidaminotransferase. Host cells may endogenously include genes encodingsuch enzymes or such genes can be recombinantly introduced. E. coli, forexample, endogenously expresses such an enzyme, IlvE (EC 2.6.1.42;GenBank accession YP_026247). In host cells where no branched-chainamino acid aminotransferase are expressed, an E. coli IlvE or any otherbranched-chain amino acid aminotransferase (e.g., IlvE from Lactococcuslactis (GenBank accession AAF34406), IlvE from Pseudomonas putida(GenBank accession NP_745648), or IlvE from Streptomyces coelicolor(GenBank accession NP_629657)), can be introduced.

In another embodiment, the production of alpha-keto acids can beachieved using the methods described in Park et al., PNAS, 104:7797-7802(2007) and Atsumi et al., Nature, 451: 86-89 (2008). For example,2-ketoisovalerate can be produced by overexpressing the genes encodingIlvI, IlvH, IlvH mutant, IlvB, IlvN, IlvGM, IlvC, or IlvD.Alternatively, 2-keto-3-methyl-valerate can be produced byoverexpressing the genes encoding IlvA and IlvI, IlvH (or AlsS ofBacillus subtilis), IlvC, IlvD, or their homologs.2-keto-4-methyl-pentanoate can also be produced by overexpressing thegenes encoding IlvI, IlvH, IlvC, IlvD and LeuA, LeuB, LeuC, LeuD, ortheir homologs.

In another example, isobutyryl-CoA can be made in a host cell, forexample in E. coli, through the coexpression of a crotonyl-CoA reductase(Ccr, EC 1.6.5.5, 1.1.1.1) and isobutyryl-CoA mutase (large subunitIcmA, EC 5.4.99.2; small subunit IcmB, EC 5.4.99.2) (Han and Reynolds,J. Bacteriol., 179: 5157 (1997)). Crotonyl-CoA is an intermediate infatty acid biosynthesis in E. coli and other microorganisms.Non-limiting examples of ccr and icm genes from selected microorganismsare listed in Table 9.

TABLE 9 ccr and icm Genes from Selected Microorganisms Organism GeneGenBank Accession # Streptomyces coelicolor Ccr NP_630556 icmA NP_629554icmB NP_630904 Streptomyces Ccr AAD53915 cinnamonensis icmA AAC08713icmB AJ246005

Formation of Branched Cyclic Fatty Alcohols and Derivatives

Branched cyclic fatty alcohols can be produced from suitable alpha ketoacids using branched cyclic fatty acid derivatives such as a branchedcyclic acyl-ACP as substrates. To produce branched cyclic fatty acidderivative substrates, genes that provide cyclic precursors (e.g., theans, chc, and plm gene families) can be introduced into a host cell andexpressed to allow initiation of fatty acid biosynthesis from branchedcyclic precursors. For example, to convert a host cell, such as E. coli,into one capable of synthesizing ω-cyclic fatty acids (cyFA), a genethat provides the cyclic precursor cyclohexylcarbonyl-CoA (CHC-CoA)(Cropp et al., Nature Biotech., 18: 980-983 (2000)) can be introducedand expressed in the host cell. Non-limiting examples of genes thatprovide CHC-CoA in E. coli include: ansJ, ansK, ansL, chcA, and ansMfrom the ansatrienin gene cluster of Streptomyces collinus (Chen et al.,Eur. J. Biochem., 261: 98-107 (1999)) or plmJ, plmK, plmL, chcA, andplmM from the phoslactomycin B gene cluster of Streptomyces sp. HK803(Palaniappan et al., J. Biol. Chem., 278: 35552-35557 (2003)) togetherwith the chcB gene (Patton et al., Biochem., 39: 7595-7604 (2000)) fromS. collinus, S. avermitilis, or S. coelicolor (see Table 10). The geneslisted in Table 10 can then be expressed to allow initiation andelongation of ω-cyclic fatty acids. Alternatively, the homologous genescan be isolated from microorganisms that make cyFA and expressed in ahost cell (e.g., E. coli).

TABLE 10 Genes for the Synthesis of CHC-CoA Organism Gene GenBankAccession No. Streptomyces collinus ansJK U72144* ansL AF268489 chcAansM chcB Streptomyces sp. HK803 pmlJK AAQ84158 pmlL AAQ84159 chcAAAQ84160 pmlM AAQ84161 Streptomyces coelicolor chcB/caiD NP_629292Streptomyces avermitilis chcB/caiD NP_629292 *Only chcA is annotated inGenBank entry U72144; ansJKLM are according to Chen et al. (Eur. J.Biochem., 261: 98-107 (1999)).

Genes fabH, acp, and fabF allow initiation and elongation of ω-cyclicfatty acids because they have broad substrate specificity. If thecoexpression of any of these genes with the genes listed in Table 10does not yield cyFA, then fabH, acp, and/or fabF homologs frommicroorganisms that make cyFAs (e.g., those listed in Table 11) can beisolated (e.g., by using degenerate PCR primers or heterologous DNAsequence probes) and coexpressed.

TABLE 11 Non-Limiting Examples of Microorganisms Containing ω-cyclicFatty Acids Organism Reference Curtobacterium pusillum ATCC19096Alicyclobacillus acidoterrestris ATCC49025 Alicyclobacillusacidocaldarius ATCC27009 Alicyclobacillus cycloheptanicus * Moore, J.Org. Chem., 62: 2173 (1997) * Uses cycloheptylcarbonyl-CoA and notcyclohexylcarbonyl-CoA as precursor for cyFA biosynthesis.

Branched Fatty Alcohol Saturation Levels

The degree of saturation in branched fatty acid derivative substrates,such as, for example, a branched acyl-ACP, (which can then be convertedinto branched fatty aldehydes and then branched fatty alcohols asdescribed herein) can be controlled by regulating the degree ofsaturation of fatty acid intermediates. For example, the sfa, gns, andfab families of genes can be expressed or overexpressed to control thesaturation of a branched acyl-ACP. In certain embodiments, the hostcells can be engineered to reduce the expression of an sfa, gns, or fabgene and control the level of saturated substrates vs. unsaturatedsubstrates, which in turn affects the production level of saturatedbranched fatty alcohols or derivatives vs. unsaturated branched fattyalcohols or derivatives.

In some instances, a host cell can be engineered to express anattenuated level of a dehydratase/isomerase and/or a ketoacyl-ACPsynthase. For example, a host cell can be engineered to express adecreased level of fabA and/or fabB. In some instances, the host cellcan be cultured or grown in the presence of unsaturated fatty acids. Insome instances, the host cell can be engineered to express oroverexpress a gene encoding a desaturases enzyme. One non-limitingexample of a desaturases is B. subtiis DesA (AF037430). Other genesencoding desaturases are known in the art can be introduced or used inthe host cell and methods described herein, such as desaturases that useacyl-ACPs, including, for example, hexadecanoyl-ACP or octadecanoyl-ACP.

In some embodiments, those cells can be engineered to produceunsaturated fatty acids by engineering the production host tooverexpress fabB or by growing the production host at low temperatures(e.g., less than 37° C.). FabB has preference to cis-δ3decenoyl-ACP andresults in unsaturated fatty acid production in E. coli. Overexpressionof fabB results in the production of a significant percentage ofunsaturated fatty acids (de Mendoza et al., J. Biol. Chem., 258:2098-2101 (1983)). The gene fabB may be inserted into and expressed inhost cells not naturally having the gene. These unsaturated fatty acidscan then be used as intermediates in host cells that are engineered toproduce branched and unsaturated fatty acid derivative substrates, suchas branched and unsaturated fatty aldehydes, which can in turn beconverted into branched and unsaturated fatty alcohols and derivatives.

In other instances, a repressor of fatty acid biosynthesis, for example,fabR (GenBank accession NP_418398), can be deleted, which will alsoresult in increased unsaturated fatty acid production in E. coli (Zhanget al., J. Biol. Chem., 277: 15558 (2002)). Similar deletions may bemade in other host cells. A further increase in unsaturated fatty acidsmay be achieved, for example, by overexpressing fabM (trans-2,cis-3-decenoyl-ACP isomerase, GenBank accession DAA05501) and controlledexpression of fabK (trans-2-enoyl-ACP reductase II, GenBank accessionNP_357969) from Streptococcus pneumoniae (Marrakchi et al., J. Biol.Chem., 277: 44809 (2002)), while deleting E. coli fabI(trans-2-enoyl-ACP reductase, GenBank accession NP_415804). In someexamples, the endogenous fabF gene can be attenuated, thus increasingthe percentage of palmitoleate (C16:1) produced.

Production of Genetic Variants

Variants can be naturally occurring or created in vitro. In particular,such variants can be created using genetic engineering techniques, suchas site directed mutagenesis, random chemical mutagenesis, ExonucleaseIII deletion procedures, or standard cloning techniques. Alternatively,such variants, fragments, analogs, or derivatives can be created usingchemical synthesis or modification procedures.

Methods of making variants are well known in the art. These includeprocedures in which nucleic acid sequences obtained from naturalisolates are modified to generate nucleic acids that encode polypeptideshaving characteristics that enhance their value in industrial orlaboratory applications. In such procedures, a large number of variantsequences having one or more nucleotide differences with respect to thesequence obtained from the natural isolate are generated andcharacterized. Typically, these nucleotide differences result in aminoacid changes with respect to the polypeptides encoded by the nucleicacids from the natural isolates.

For example, variants can be created using error prone PCR (see, e.g.,Leung et al., Technique, 1: 11-15 (1989); and Caldwell et al., PCRMethods Applic., 2: 28-33 (1992)). In error prone PCR, PCR is performedunder conditions where the copying fidelity of the DNA polymerase islow, such that a high rate of point mutations is obtained along theentire length of the PCR product. Briefly, in such procedures, nucleicacids to be mutagenized (e.g., a fatty aldehyde biosyntheticpolynucleotide sequence) are mixed with PCR primers, reaction buffer,MgCl₂, MnCl₂, Taq polymerase, and an appropriate concentration of dNTPsfor achieving a high rate of point mutation along the entire length ofthe PCR product. For example, the reaction can be performed using 20fmoles of nucleic acid to be mutagenized (e.g., a fatty aldehydebiosynthetic polynucleotide sequence), 30 pmole of each PCR primer, areaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3), and 0.01%gelatin, 7 mM MgCl₂, 0.5 mM MnCl₂, 5 units of Taq polymerase, 0.2 mMdGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR can be performed for 30cycles of 94° C. for 1 min, 45° C. for 1 min, and 72° C. for 1 min.However, it will be appreciated that these parameters can be varied asappropriate. The mutagenized nucleic acids are then cloned into anappropriate vector and the activities of the polypeptides encoded by themutagenized nucleic acids are evaluated.

Variants can also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described in, for example, Reidhaar-Olsonet al., Science, 241: 53-57 (1988).

Variants can also be generated by assembly PCR, which involves theassembly of a PCR product from a mixture of small DNA fragments. A largenumber of different PCR reactions occur in parallel in the same vial,with the products of one reaction priming the products of anotherreaction. Assembly PCR is described in, e.g., U.S. Pat. No. 5,965,408.

Still another method of generating variants is sexual PCR mutagenesis,wherein forced homologous recombination occurs between DNA molecules ofdifferent, but highly related, DNA sequence in vitro as a result ofrandom fragmentation of the DNA molecule based on sequence homology.This is followed by fixation of the crossover by primer extension in aPCR reaction. Sexual PCR mutagenesis is described in, for example,Stemmer, Proc. Natl. Acad. Sci. USA, 91: 10747-10751 (1994).

Variants can also be created by in vivo mutagenesis. In someembodiments, random mutations in a nucleic acid sequence are generatedby propagating the sequence in a bacterial strain, such as an E. colistrain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type strain. Propagating a DNA sequence (e.g., a BKDpolynucleotide sequence, a beta acyl-ACP synthase polynucleotidesequence, a fatty aldehyde biosynthesis polynucleotide sequence, or afatty alcohol biosynthesis polynucleotide sequence) in one of thesestrains will eventually generate random mutations within the DNA.Mutator strains suitable for use for in vivo mutagenesis are describedin, for example, International Publication WO 91/016427.

Variants can also be generated using cassette mutagenesis. In cassettemutagenesis, a small region of a double stranded DNA molecule isreplaced with a synthetic oligonucleotide “cassette” that differs fromthe native sequence. The oligonucleotide often contains a completelyand/or partially randomized native sequence.

Recursive ensemble mutagenesis can also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(i.e., protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described in, for example, Arkin et al., Proc. Natl.Acad. Sci. USA, 89: 7811-7815 (1992).

In some embodiments, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is describedin, for example, Delegrave et al., Biotech. Res., 11: 1548-1552 (1993).Random and site-directed mutagenesis are described in, for example,Arnold, Curr. Opin. Biotech., 4: 450-455 (1993).

In some embodiments, variants are created using shuffling procedureswherein portions of a plurality of nucleic acids that encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences that encode chimeric polypeptides as described in, forexample, U.S. Pat. Nos. 5,965,408 and 5,939,250.

Polynucleotide variants also include nucleic acid analogs. Nucleic acidanalogs can be modified at the base moiety, sugar moiety, or phosphatebackbone to improve, for example, stability, hybridization, orsolubility of the nucleic acid. Modifications at the base moiety includedeoxyuridine for deoxythymidine and 5-methyl-2′-deoxycytidine or5-bromo-2′-doxycytidine for deoxycytidine. Modifications of the sugarmoiety include modification of the 2′ hydroxyl of the ribose sugar toform 2′-halo, 2′-O-methyl or 2′-O-allyl sugars. The deoxyribosephosphate backbone can be modified to produce morpholino nucleic acids,in which each base moiety is linked to a six-membered, morpholino ring,or peptide nucleic acids, in which the deoxyphosphate backbone isreplaced by a pseudopeptide backbone and the four bases are retained.(See, e.g., Summerton et al., Antisense Nucleic Acid Drug Dev., 7:187-195 (1997); and Hyrup et al., Bioorgan. Med. Chem., 4: 5-23 (1996)).In addition, the deoxyphosphate backbone can be replaced with, forexample, a phosphorothioate or phosphorodithioate backbone, aphosphoroamidite, or an alkyl phosphotriester backbone.

Production of Polypeptide Variants

Conservative substitutions are those that substitute an amino acid in apolypeptide by another amino acid of similar characteristics. Commonconservative substitutions include, without limitation: replacing analiphatic amino acid, such as alanine, valine, leucine, and isoleucine,with another aliphatic amino acid; replacing a serine with a threonineor vice versa; replacing an acidic residue, such as aspartic acid andglutamic acid, with another acidic residue; replacing a residue bearingan amide group, such as asparagine and glutamine, with another residuebearing an amide group; replacing a basic residue, such as lysine andarginine, with another basic residue; and replacing an aromatic residue,such as phenylalanine and tyrosine, with another aromatic residue.

Other polypeptide variants are those in which one or more amino acidresidues include a substituent group. Still other polypeptide variantsare those in which the polypeptide is associated with another compound,such as a compound to increase the half-life of the polypeptide (e.g.,polyethylene glycol).

Additional polypeptide variants are those in which additional aminoacids are fused to the polypeptide, such as a leader sequence, asecretory sequence, a proprotein sequence, or a sequence whichfacilitates purification, enrichment, or stabilization of thepolypeptide.

In some instances, the polypeptide variants described herein retain thesame biological function as a polypeptide from which they are derived(e.g., retain branched-chain alpha keto acid dehydrogenase activity,retain beta ketoyacyl ACP synthase activity, such as FabH activity, orretain fatty aldehyde biosynthetic activity, such as carboxylic acid orfatty acid reductase activity) and have amino acid sequencessubstantially identical thereto.

In other instances, the polypeptide variants have at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or more than about 95% homology toan amino acid sequence from which they are derived. In anotherembodiment, the polypeptide variants include a fragment comprising atleast about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof.

The polypeptide variants or fragments thereof can be obtained byisolating nucleic acids encoding them using techniques described hereinor by expressing synthetic nucleic acids encoding them. Alternatively,polypeptide variants or fragments thereof can be obtained throughbiochemical enrichment or purification procedures. The sequence ofpolypeptide variants or fragments can be determined by proteolyticdigestion, gel electrophoresis, and/or microsequencing. The sequence ofthe polypeptide variants or fragments can then be compared to the aminoacid sequence from which it is derived using any of the programsdescribed herein.

The polypeptide variants and fragments thereof can be assayed forenzymatic activity. For example, the polypeptide variants or fragmentscan be contacted with a substrate under conditions that allow thepolypeptide variants or fragments to function. A decrease in the levelof the substrate or an increase in the level of the desired product canbe measured to determine its activity.

Modifications to Increase Conversion of Branched Substrates to BranchedFatty Alcohol

Host cells can be engineered using known polypeptides to producebranched fatty alcohols from branched substrate, including, for example,a branched fatty acid, a branched fatty acid derivative, a branchedacyl-CoA, or a branched acyl-CoA derivative substrate. For example, onemethod of making branched fatty alcohols involves increasing theexpression of, or expressing more active forms of, fatty alcohol formingacyl-CoA reductases (encode by a gene such as acr1 from FAR, EC1.2.1.50/1.1.1) or acyl-CoA reductases (EC 1.2.1.50) and alcoholdehydrogenase (EC 1.1.1.1).

The host cell can also be, for example, modified or engineered, suchthat it expresses or overexpresses at least one (E1) subunit of abranched-chain alpha keto acid dehydrogenase complex, and a betaketoacyl-ACP synthase. The host cell can be further engineered such thatit expresses or overexpresses a fatty aldehyde biosynthesis polypeptideand/or a fatty alcohol biosynthesis polypeptide. Alternatively, the hostcell can be engineered such that it expresses or overexpresses anacyl-ACP reductase polypeptide and a fatty alcohol biosynthesispolypeptide.

In certain embodiments, the gene encoding the subunits of branched-chainalpha keto acid dehydrogenase complex can be derived from a bacterium, aplant, an insect, a yeast, a fungus, or a mammal. For example, thesubunits of the branched-chain alpha keto acid dehydrogenase complex canbe derived from a bacterium that uses branched amino acids as carbonsource, including, for example, Pseudomonas putida or Bacillus subtilis.In another example, the branched-chain alpha-keto acid dehydrogenasecomplex polypeptide can be from a bacterium that comprises branchedfatty acids in its phospholipids, including, e.g., a Legionella,Stenotrophomonas, Alteromonas, Flavobacterium, Myxococcus, Bccteroides,Micrococcus, Staphylococcus, Bacillus, Clostridium, Listeria,Lactococcus, or Streptomyces. In some embodiments, the bacterium is aLeginella pneumophila, Stenotrophomonas maltophilia, Alteromonasmacleodii, Flabobacterium phsychrophilum, Myxococcus Xanthus,Bacteroides thetaiotaomicron, Macrococcus luteus, Staphylococcus aureus,Clostridium thermocellum, Listeria monocytogenes, Streptomyces lividans,Streptomyces coelicolor, Streptomyces glaucescens, Streptococcuspneumoniae, Streptomyces peucetius, Streptococcus pyogenes, Escherichiacoli, Escherichia coli K-12, Lactococcus lactis ssp. Lactis,Mycobacterium tuberculosis, Enterococcus tuberculosis, Bacillussubtilis, Lactobacillus plantarum. In some embodiments, suitable fattyaldehyde biosynthesis polypeptides, fatty alcohol biosynthesispolypeptides, acyl-ACP reductases, and other polypeptides of theinvention can be from a mycobacterium selected from Mycobacteriumsmegmatis, Mycobacterium abscessus, Mycobacterium avium, Mycobacteriumbovis, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacteriummarinum, or Mycobacterium ulcerans. In other embodiments, the bacteriumis Nocardia sp. NRRL 5646, Nocardia farcinica, Streptomyces griseus,Salinispora arenicola, or Clavibacter michiganenesis. In yet furtherembodiments, the polypeptide of the invention is derived from acyanobacterium, including, for example, Synechococcus elongatus PCC7942,Synechocystis sp. PCC6803, Cyanothece sp. ATCC51142, Prochlorococcusmarinus subsp. pastoris str. CCMP1986 PMM0533, Gloeobacter violaceusPCC7421, Nostoc punctiforme PCC73102, Anabaena variabilis ATCC29413,Synechococcus elongatus PCC6301, and Nostoc sp. PCC 7120, Microcoleuschthonoplastes PCC7420, Arthrospira maxima CS-328, Lyngbya sp. PCC8106,Nodularia spumigena CCY9414, Trichodesmium erythraeum IMS101,Microcystis aeruginosa, Nostoc azollae, Anabaena variabilis, Crocophaerawatsonii, Thermosynechococcus elongatus, Gloeobacer violaceus,Cyanobium, or Prochlorococcus marinus.

Genetic Engineering of Host Cells to Produce Branched Fatty Alcohols

Various host cells can be used to produce branched fatty alcohols, asdescribed herein. A host cell can be any prokaryotic or eukaryotic cell.For example, the host cell can be bacterial cells (such as E. coli),insect cells, yeast, or mammalian cells (such as Chinese hamster ovarycells (CHO) cells, COS cells, VERO cells, BHK cells, HeLa cells, Cv1cells, MDCK cells, 293 cells, 3T3 cells, or PC12 cells). Other exemplaryhost cells include cells from the members of the genus Escherichia,Bacillus, Lactobacillus, Rhodococcus, Pseudomonas, Aspergillus,Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces,Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus,Trametes, Chrysosporium, Saccharomyces, Schizosaccharomyces, Yarrowia,or Streptomyces. Yet other exemplary host cells can be a Bacillus lentuscell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, aBacillus licheniformis cell, a Bacillus alkalophilus cell, a Bacilluscoagulans cell, a Bacillus circulans cell, a Bacillus pumilis cell, aBacillus thuringiensis cell, a Bacillus clausii cell, a Bacillusmegaterium cell, a Bacillus subtilis cell, a Bacillus amyloliquefacienscell, a Trichoderma koningii cell, a Trichoderma viride cell, aTrichoderma reesei cell, a Trichoderma longibrachiatum cell, anAspergillus awamori cell, an Aspergillus fumigates cell, an Aspergillusfoetidus cell, an Aspergillus nidulans cell, an Aspergillus niger cell,an Aspergillus oryzae cell, a Humicola insolens cell, a Humicolalanuginose cell, a Rhizomucor miehei cell, a Mucor michei cell, aStreptomyces lividans cell, a Streptomyces murinus cell, or anActinomycetes cell. Host cells can also be cyanobacterial cells such as,for example, Synechoccus sp., Synechoccus elongatus, or Synechocystissp. cells.

In a preferred embodiment, the host cell is an E. coli cell, aSaccharomyces cerevisiae cell, or a Bacillus subtilis cell. For example,the host cell can be one from E. coli strain B, C, K, or W. Othersuitable host cells are known to those skilled in the art.

Various methods well known in the art can be used to geneticallyengineer host cells to produce branched fatty alcohols. The methods caninclude the use of vectors, preferably expression vectors, containing anucleic acid encoding the first (E1 alpha/beta) subunit of abranched-chain alpha keto acid dehydrogenase, and optionally also thesecond (E2) and/or the third (E3) subunit of that enzyme, and/or a betaketoacyl-ACP synthase, and/or a fatty aldehyde biosynthetic polypeptide,and/or an alcohol dehydrogenase, and/or an acyl-ACP reductases,described herein, polypeptide variant, or a fragment thereof. Thoseskilled in the art will appreciate a variety of viral vectors (forexample, retroviral vectors, lentiviral vectors, adenoviral vectors, andadeno-associated viral vectors) and non-viral vectors can be used in themethods described herein.

The recombinant expression vectors can include polynucleotides describedherein in a form suitable for expression in a host cell. The recombinantexpression vectors can include one or more control sequences, selectedon the basis of the host cell to be used for expression. The controlsequence is operably linked to the nucleic acid sequence to beexpressed. Such control sequences are described, for example, inGoeddel, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Control sequences include those thatdirect constitutive expression of a nucleotide sequence in many types ofhost cells and those that direct expression of the nucleotide sequenceonly in certain host cells (e.g., tissue-specific regulatory sequences).It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. The expression vectors described herein can be introduced into hostcells to produce polypeptides, including fusion polypeptides, encoded bythe nucleic acids as described herein.

In some embodiments, recombinant expression vectors can be designed forexpression of a gene encoding a first (E1 alpha/beta) subunit, andoptionally a second (E2) and/or a third (E3) subunit of a branched-chainalpha-keto acid dehydrogenase (or variant) and/or a gene encoding abeta-ketoacyl ACP synthase (or variant), and/or a gene encoding a fattyaldehyde biosynthesis polypeptide (or variant), and/or a gene encodingan alcohol dehydrogenase (or variant), and/or a gene encoding anacyl-ACP reductases (or variant) in a suitable host cell. Suitable hostcells are discussed further in Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example, by using T7 promoter regulatorysequences and T7 polymerase.

Expression of genes encoding polypeptides in prokaryotes, for example,E. coli, is most often carried out with vectors containing constitutiveor inducible promoters directing the expression of either fusion ornon-fusion polypeptides. Fusion vectors add a number of amino acids to apolypeptide encoded therein, usually to the amino terminus of therecombinant polypeptide. Such fusion vectors typically serve threepurposes: (1) to increase expression of the recombinant polypeptide; (2)to increase the solubility of the recombinant polypeptide; and (3) toaid in the purification of the recombinant polypeptide by acting as aligand in affinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant polypeptide. This enables separation of therecombinant polypeptide from the fusion moiety after purification of thefusion polypeptide. Examples of such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin, and enterokinase.Exemplary fusion expression vectors include pGEX (Pharmacia BiotechInc.; Smith et al., Gene, 67: 31-40 (1988)), pMAL (New England Biolabs,Beverly, Mass.), and pRITS (Pharmacia, Piscataway, N.J.), which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant polypeptide.

Examples of inducible, non-fusion E. coli expression vectors includepTrc (Amann et al., Gene, 69: 301-315 (1988)) and pET 11d (Studier etal., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990), pp. 60-89). Target gene expression fromthe pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident λ, prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

One strategy to maximize expression is to express the polypeptide in ahost cell with an impaired capacity to proteolytically cleave therecombinant polypeptide (see Gottesman, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), pp.119-128). Another strategy is to alter the nucleic acid sequence to beinserted into an expression vector so that the individual codons foreach amino acid are those preferentially utilized in the host cell (Wadaet al., Nucleic Acids Res., 20: 2111-18 (1992)). These strategies can becarried out by standard DNA synthesis techniques.

In another embodiment, the host cell is a yeast cell, and the expressionvector is a yeast expression vector. Examples of vectors for expressionin yeast S. cerevisiae include pYepSecl (Baldari et al., EMBO J., 6:229-234 (1987)), pMFa (Kurjan et al., Cell, 30: 933-943 (1982)), pJRY88(Schultz et al., Gene, 54: 113-123 (1987)), pYES2 (InvitrogenCorporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego,Calif.).

Alternatively, polypeptides described herein can be expressed in insectcells using baculovirus expression vectors. Available baculovirusvectors include, for example, the pAc series (Smith et al., Mol. CellBiol., 3: 2156-2165 (1983)) and the pVL series (Lucklow et al.,Virology, 170: 31-39 (1989)).

In yet another embodiment, the polypeptides described herein can beexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, Nature,329: 840 (1987)) and pMT2PC (Kaufman et al., EMBO J., 6: 187-195(1987)). When used in mammalian cells, the expression vector's controlfunctions can be provided by viral regulatory elements. Commonly usedpromoters include those derived from polyoma, Adenovirus 2,cytomegalovirus, and Simian Virus 40. Other suitable expression systemsfor both prokaryotic and eukaryotic cells are described in chapters16-17 of Sambrook et al., eds., Molecular Cloning: A Laboratory Manual.2^(nd), ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N. Y., 1989.

Vectors can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” refer to a variety ofart-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation.

It is known that, depending upon the expression vector andtransformation technique used, only a small fraction of bacterial cellswill take-up and replicate the expression vector. In order to identifyand select these transformants, a gene that encodes a selectable marker(e.g., resistance to antibiotics) can be introduced into the host cellsalong with the gene of interest. Selectable markers include those thatconfer resistance to drugs, such as ampicillin, kanamycin,chloramphenicol, or tetracycline. Nucleic acids encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding a polypeptide described herein or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

It is known that, depending upon the expression vector and transfectiontechnique used, only a small fraction of mammalian cells may integratethe foreign DNA into their genome. In order to identify and select theseintegrants, a gene that encodes a selectable marker (e.g., resistance toantibiotics) can be introduced into the host cells along with the geneof interest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin, and methotrexate. Nucleicacids encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding a polypeptide described herein or canbe introduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

Transport Proteins

Transport proteins can export or excrete polypeptides and organiccompounds (e.g., branched fatty alcohols) out of a host cell. A numberof transport and efflux proteins can be modified to selectively secreteparticular types of compounds such as branched fatty alcohols.

Non-limiting examples of suitable transport proteins are ATP-BindingCassette (ABC) transport proteins, efflux proteins, and fatty acidtransporter proteins (FATP). Additional suitable transport proteinsinclude the ABC transport proteins from organisms such as Caenorhabditiselegans, Arabidopsis thalania, Alkaligenes eutrophus, or Rhodococcuserythropolis. Exemplary ABC transport proteins include, withoutlimitation, CERS, AtMRPS, AmiS2, and AtPGP1. Host cells can also bechosen for their endogenous ability to secrete organic compounds. Theefficiency of organic compound production and secretion into the hostcell environment (e.g., culture medium, fermentation broth) can beexpressed as a ratio of intracellular product to extracellular product.For example, the ratio can be about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3,1:4, or 1:5.

Fermentation

The production and isolation of branched fatty alcohols can be enhancedby employing beneficial fermentation techniques. One method formaximizing production while reducing costs is increasing the percentageof the carbon source that is converted to the branched fatty alcoholproducts.

During normal cellular lifecycles, carbon is used in cellular functionssuch as producing lipids, saccharides, proteins, organic acids, andnucleic acids. Reducing the amount of carbon necessary forgrowth-related activities can increase the efficiency of carbon sourceconversion to product. This can be achieved by, for example, firstgrowing host cells to a desired density (for example, a density achievedat the peak of the log phase of growth). At such a point, replicationcheckpoint genes can be harnessed to stop the growth of cells.Specifically, quorum sensing mechanisms (reviewed in Camilli et al.,Science, 311: 1113 (2006); Venturi FEMS Microbio. Rev., 30: 274-291(2006); and Reading et al., FEMS Microbiol. Lett., 254: 1-11 (2006)) canbe used to activate checkpoint genes, such as p53, p21, or othercheckpoint genes.

Genes that can be activated to stop cell replication and/or growth in E.coli include umuDC genes. The overexpression of umuDC genes stops theprogression from stationary phase to exponential growth (Murli et al.,J. Bact., 182: 1127 (2000)). UmuC is a DNA polymerase that can carry outtranslesion synthesis over non-coding lesions—the mechanistic basis ofmost UV and chemical mutagenesis. The umuDC gene products are involvedin the process of translesion synthesis and also serve as a DNA sequencedamage checkpoint. The umuDC gene products include UmuC, UmuD, umuD′,UmuD′₂C, UmuD′ ₂, and UmuD₂. Simultaneously, product-producing genes canbe activated, thus minimizing the need for replication and maintenancepathways to be used while a fatty aldehyde is being made. Host cells canalso be engineered to express umuC and umuD from E. coli in pBAD24 underthe prpBCDE promoter system through de novo synthesis of this gene withthe appropriate end-product production genes.

The percentage of input carbons converted to branched fatty alcohols canbe a cost driver. The more efficient the process is (i.e., the higherthe percentage of input carbons converted to branched fatty alcohols),the less expensive the process will be. For oxygen-containing carbonsources (e.g., glucose and other carbohydrate based sources), the oxygenmust be released in the form of carbon dioxide. For every 2 oxygen atomsreleased, a carbon atom is also released leading to a maximaltheoretical metabolic efficiency of approximately 34% (w/w) (for fattyacid derived products). This figure, however, changes for other organiccompounds and carbon sources. Typical efficiencies in the literature areapproximately less than 5%. Host cells engineered to produce fattyalcohols can have greater than about 1, 3, 5, 10, 15, 20, 25, and 30%efficiency. In one example, host cells can exhibit an efficiency ofabout 10% to about 25%. In other examples, such host cells can exhibitan efficiency of about 25% to about 30%. In other examples, host cellscan exhibit greater than 30% efficiency.

The host cell can be additionally engineered to express recombinantcellulosomes, such as those described in International Publication WO2008/100251. These cellulosomes can allow the host cell to usecellulosic material as a carbon source. For example, the host cell canbe additionally engineered to express invertases (EC 3.2.1.26) so thatsucrose can be used as a carbon source. Similarly, the host cell can beengineered using the teachings described in U.S. Pat. Nos. 5,000,000;5,028,539; 5,424,202; 5,482,846; and 5,602,030, so that the host cellcan assimilate carbon efficiently and use cellulosic materials as carbonsources.

In one example, the fermentation chamber can enclose a fermentation thatis undergoing a continuous reduction. In this instance, a stablereductive environment can be created. The electron balance can bemaintained by the release of carbon dioxide (in gaseous form). Effortsto augment the NAD/H and NADP/H balance can also facilitate instabilizing the electron balance. The availability of intracellularNADPH can also be enhanced by engineering the host cell to express anNADH:NADPH transhydrogenase. The expression of one or more NADH:NADPHtranshydrogenases converts the NADH produced in glycolysis to NADPH,which can enhance the production of fatty alcohols.

For small scale production, the engineered host cells can be (a) grownin batches of, for example, about 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10L, (b) fermented, and (c) induced to express desired bkd genes,beta-ketoacyl ACP synthase genes, fatty aldehyde biosynthesis genes,alcohol dehydrogenase genes, and/or acyl-ACP reductases genes, based onthe specific genes encoded in the appropriate plasmids. For large scaleproduction, the engineered host cells can be (a) grown in batches ofabout 10 L, 100 L, 1000 L, 10,000 L, 100,000 L, 1,000,000 L, or larger,(b) fermented, and (c) induced to express the desired bkd genes,beta-ketoacyl ACP synthase genes, fatty aldehyde biosynthesis genes,alcohol dehydrogenase genes, and/or acyl-ACP reductases genes based onthe specific genes encoded in the plasmids or incorporated into the hostcell's genome.

For example, a suitable production host, such as an E. coli, harboringplasmids containing the desired genes or having the genes integrated inits chromosome can be incubated in a suitable reactor, for example a 1 Lreactor, for 20 hours at 37° C. in an M9 medium supplemented with 2%glucose, carbenicillin, and chloramphenicol. When the OD₆₀₀ of theculture reaches 0.9, the production host can be induced with IPTGalcohol After incubation, the spent media can be extracted and theorganic phase can be examined for the presence of branched fattyalcohols using GC-MS.

In some instances, after the first hour of induction, aliquots of nomore than about 10% of the total cell volume can be removed each hourand allowed to sit without agitation to allow the branched fattyalcohols to rise to the surface and undergo a spontaneous phaseseparation or precipitation. The branched fatty alcohol component canthen be collected, and the aqueous phase returned to the reactionchamber. The reaction chamber can be operated continuously. When theOD₆₀₀ drops below 0.6, the cells can be replaced with a new batch grownfrom a seed culture.

Producing Branched Fatty Alcohols and Derivatives Using Cell-FreeMethods

In some embodiments, branched fatty alcohols can be produced using apurified polypeptide (e.g., a branched-chain alpha keto aciddehydrogenase complex polypeptide) described herein and a substrate(e.g., an alpha keto acid, malonyl-CoA, 2-oxo-isovalerate,2-oxo-isobutylrate, 2-oxo-3-methyl-valerate. 2-oxo-isocaproate,2-oxoglutarate, 2-oxopentanoate, 3-methyl-2-oxobutanoate,3-methyl-2-oxopentanoate, 4-methyl-2-oxopentanoate, or pyruvate)produced, for example, by a method described herein. For example, a hostcell can be engineered to express a branched-chain alpha keto aciddehydrogenase polypeptide or the E1 (alpha and beta), and optionally,the E2 and/or the E3 subunits thereof, or variants as described herein.The host cell can be cultured under conditions sufficient to allowexpression of the polypeptide. Cell free extracts can then be generatedusing known methods, including, for example, cell lysis using detergentsor sonication. The expressed polypeptides can be purified. Thereafter,substrates described herein can be added to the cell free extracts andmaintained under conditions to allow conversion of the substrates (e.g.,alpha keto acids, such as 2-oxo-isovalerate, 2-oxo-isobutylrate,2-oxo-3-methyl-valerate. 2-oxo-isocaproate, 2-oxoglutarate,2-oxopentanoate, 3-methyl-2-oxobutanoate, 3-methyl-2-oxopentanoate,4-methyl-2-oxopentanoate, or pyruvate) to branched chain acyl-CoAs,which can then be converted into branched fatty aldehydes and branchedfatty alcohols. The branched fatty alcohols can then be separated andpurified using known techniques.

Post-Production Processing

Depending on the intended use of the branched fatty alcohols produced inaccordance with the methods here, post-production processing may or maynot be necessary. As such, in certain industrial applications, theproduced branched fatty alcohols and/or derivatives may be suitably usedper se as surfactants. Moreover, such surfactants can be directlyblended or formulated into suitable cleaning compositions.

The branched fatty alcohols produced during fermentation can beseparated from the fermentation media, using any known technique forseparating fatty alcohols from aqueous media. One exemplary separationprocess is a two phase (bi-phasic) separation process, which involvesfermenting the genetically engineered host cells under conditionssufficient to produce a branched fatty alcohol, allowing it to collectin an organic phase, and separating the organic phase from the aqueousfermentation broth. This method can be practiced in both a batch andcontinuous fermentation processes.

Bi-phasic separation uses the relative immiscibility of fatty alcoholsto facilitate separation. Immiscible refers to the relative inability ofa compound to dissolve in water and is defined by the compound'spartition coefficient. One of ordinary skill in the art will appreciatethat by choosing a fermentation broth and organic phase, such that thebranched fatty alcohol being produced has a high logP value, thebranched fatty alcohol can separate into the organic phase, even at verylow concentrations, in the fermentation vessel.

The branched fatty alcohols produced by the methods described herein canbe relatively immiscible in the fermentation broth and the cytoplasm.Therefore, the branched fatty alcohol can collect in an organic phaseeither intracellularly or extracellularly. The collection of theproducts in the organic phase can lessen the impact of the branchedfatty alcohol on cellular function and can allow the host cell toproduce more product.

The branched fatty alcohol can thus be produced as a homogeneouscompounds wherein at least about 60%, 70%, 80%, 90%, or 95% of thebranched fatty alcohols produced will have carbon chain lengths thatvary by less than about 6 carbons, less than about 4 carbons, or lessthan about 2 carbons. These compounds can also be produced with arelatively uniform degree of saturation. They can be used per se assurfactants or can be formulated into suitable cleaning compositions.They can also be used as fuels, fuel additives, starting materials forproduction of other chemical compounds (e.g., polymers, surfactants,plastics, textiles, solvents, adhesives, etc.), or personal careadditives. These compounds can also be used as feedstock for subsequentreactions, for example, hydrogenation, catalytic cracking (e.g., viahydrogenation, pyrolisis, or both), and can be dehydrated to make otherproducts. In particular, these branched products confer low volatility,beneficial low-temperature properties, as well as oxidative stability,making them ideal for low temperature applications such as in householdcleaning compositions and personal and beauty care products.

In some embodiments, the branched fatty alcohols produced using methodsdescribed herein can contain between about 50% and about 90% carbon, orbetween about 5% and about 25% hydrogen. In other embodiments, thebranched fatty alcohols produced using methods described herein cancontain between about 65% and about 85% carbon, or between about 10% andabout 15% hydrogen.

In some embodiments, the branched fatty alcohols produced in accordancewith the disclosure herein comprises a C₆-C₂₆ branched fatty alcohol. Insome embodiments, the branched fatty alcohol comprises a C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃,C₂₄, C₂₅, or a C₂₆ branched fatty alcohol. In particular embodiments,the branched fatty alcohol is a C₆, C₈, C₁₀, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆,C₁₇, or C₁₈ branched fatty alcohol. In certain embodiments, the hydroxylgroup of the branched fatty alcohol is in the primary (C₁) position. Incertain embodiment, the branched fatty alcohol is an iso-fatty alcoholor an anteiso-fatty alcohol. In exemplary embodiments, the branchedfatty alcohol is selected from iso-C_(7:0), iso-C_(8:0), iso-C_(9:0),iso-C_(10:0), iso-C_(11:0), iso-C_(12:0), iso-C_(13:0), iso-C_(14:0),iso-C_(15:0), iso-C_(16:0), iso-C_(17:0), iso-C_(18:0), iso-C_(19:0),anteiso-C_(7:0), anteiso-C_(8:0), anteiso-C_(9:0), anteiso-C_(10:0),anteiso-C_(11:0), anteiso-C_(12:0), anteiso-C_(13:0), anteiso-C_(14:0),anteiso-C_(15:0), anteiso-C_(16:0), anteiso-C_(17:0), anteiso-C_(18:0),and anteiso-C_(19:0) fatty alcohol.

In certain embodiments, the fatty alcohol product can comprise straightchain fatty alcohols. In other embodiments, the branched fatty alcoholsproduced by the host cells described herein can comprise one or morepoints of branching. In certain embodiments, the branched fatty alcoholsproduced by the host cells as described herein can comprise one or morecyclic moieties.

In some embodiments, the branched fatty alcohols can be unsaturatedbranched fatty alcohols. For example, the branched fatty alcoholsproduced in accordance with the present description can bemonounsaturated branched fatty alcohols. In certain embodiments, theunsaturated branched fatty alcohol can be a C6:1, C7:1, C8:1, C9:1,C10:1, C11:1, C12:1, C13:1, C14:1, C15:1, C16:1, C17:1, C18:1, C19:1,C20:1, C21:1, C22:1, C23:1, C24:1, C25:1, or a C26:1 unsaturatedbranched fatty alcohol. In other embodiments, the branched fatty alcoholis unsaturated at the omega-7 position. In certain embodiments, theunsaturated branched fatty alcohol comprises a cis double bond.

In some embodiments, branched fatty alcohols are produced at a relativeyield to a straight-chain fatty alcohol at about 20%, for example, atabout 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or higher. In an exemplaryembodiment, the total amount of branched fatty alcohols produced isestimated to about 45% to about 50% relative to the amount ofstraight-chain fatty alcohols produced by a host cell.

In any of the aspects described herein, the production yield of fattyalcohols, including branched fatty alcohols and straight chain fattyalcohol, is about 1 mg/L, 5 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, 25 mg/L,about 50 mg/L, about 75 mg/L, about 100 mg/L, about 125 mg/L, about 150mg/L, about 175 mg/L, about 200 mg/L, about 225 mg/L, about 250 mg/L,about 275 mg/L, about 300 mg/L, about 325 mg/L, about 350 mg/L, about375 mg/L, about 400 mg/L, about 425 mg/L, about 450 mg/L, about 475mg/L, about 500 mg/L, about 525 mg/L, about 550 mg/L, about 575 mg/L,about 600 mg/L, about 625 mg/L, about 650 mg/L, about 675 mg/L, about700 mg/L, about 725 mg/L, about 750 mg/L, about 775 mg/L, about 800mg/L, about 825 mg/L, about 850 mg/L, about 875 mg/L, about 900 mg/L,about 925 mg/L, about 950 mg/L, about 975 mg/L, about 1000 g/L, about1050 mg/L, about 1075 mg/L, about 1100 mg/L, about 1125 mg/L, about 1150mg/L, about 1175 mg/L, about 1200 mg/L, about 1225 mg/L, about 1250mg/L, about 1275 mg/L, about 1300 mg/L, about 1325 mg/L, about 1350mg/L, about 1375 mg/L, about 1400 mg/L, about 1425 mg/L, about 1450mg/L, about 1475 mg/L, about 1500 mg/L, about 1525 mg/L, about 1550mg/L, about 1575 mg/L, about 1600 mg/L, about 1625 mg/L, about 1650mg/L, about 1675 mg/L, about 1700 mg/L, about 1725 mg/L, about 1750mg/L, about 1775 mg/L, about 1800 mg/L, about 1825 mg/L, about 1850mg/L, about 1875 mg/L, about 1900 mg/L, about 1925 mg/L, about 1950mg/L, about 1975 mg/L, about 2000 mg/L, or more.

In another aspect, the branched fatty alcohol produced in accordancewith the present invention is produced by culturing a host celldescribed herein in a medium having a low level of iron, underconditions sufficient to produce a branched fatty alcohol. In particularembodiments, the medium contains less than about 500 μM iron, less thanabout 400 μM iron, less than about 300 μM iron, less than about 200 μMiron, less than about 150 μM iron, less than about 100 μM iron, lessthan about 90 μM iron, less than about 80 μM iron, less than about 70 μMiron, less than about 60 μM iron, less than about 50 μM iron, less thanabout 40 μM iron, less than about 30 μM iron, less than about 20 μMiron, less than about 10 μM iron, or less than about 5 μM iron. Incertain embodiments, the medium does not contain iron.

Bioproducts (e.g., surfactants and cleaning compositions) comprisingmicrobially produced branched fatty alcohols and/or derivatives,produced using the fatty acid biosynthetic pathway, have not beenproduced from renewable sources and, as such, are new compositions ofmatter. These new bioproducts can be distinguished from organiccompounds derived from petrochemical carbon on the basis of dualcarbon-isotopic fingerprinting or ¹⁴C dating. Additionally, the specificsource of biosourced carbon (e.g., glucose vs. glycerol) can bedetermined by dual carbon-isotopic fingerprinting (see, e.g., U.S. Pat.No. 7,169,588, which is herein incorporated by reference).

The ability to distinguish bioproducts from petroleum based organiccompounds is beneficial in tracking these materials in commerce. Organiccompounds or chemicals comprising both biologically based and petroleumbased carbon isotope profiles may be distinguished from organiccompounds and chemicals made only of petroleum based materials. Hence,the surfactants and cleaning compositions of the present invention befollowed in commerce on the basis of their unique carbon isotopeprofile.

Surfactants or cleaning compositions produced in accordance with thepresent disclosure can be distinguished from petroleum-derived compoundsby comparing the stable carbon isotope ratio (¹³C/¹²C) of each. The¹³C/¹²C ratio in a given bioproduct is a consequence of the ¹³C/¹²Cratio in atmospheric carbon dioxide at the time the carbon dioxide isfixed. It also reflects the precise metabolic pathway. Regionalvariations also occur. Petroleum, C₃ plants (the broadleaf), C₄ plants(the grasses), and marine carbonates all show significant differences in¹³C/¹²C and the corresponding δ¹³C values. Moreover, lipid matter of C₃and C₄ plants analyze differently than materials derived from thecarbohydrate components of the same plants as a consequence of themetabolic pathway.

Within the precision of measurement, ¹³C shows large variations due toisotopic fractionation effects, the most significant of which forbioproducts is the photosynthetic mechanism. The major cause ofdifferences in the carbon isotope ratio in plants is closely associatedwith differences in the pathway of photosynthetic carbon metabolism inthe plants, particularly the reaction occurring during the primarycarboxylation (i.e., the initial fixation of atmospheric CO₂). Two largeclasses of vegetation are those that incorporate the “C₃” (orCalvin-Benson) photosynthetic cycle and those that incorporate the “C₄”(or Hatch-Slack) photosynthetic cycle.

In C₃ plants, the primary CO₂ fixation/carboxylation reaction involvesthe enzyme ribulose-1,5-diphosphate carboxylase, and the first stableproduct is a 3-carbon compound. C₃ plants, such as hardwoods andconifers, are dominant in the temperate climate zones.

In C₄ plants, an additional carboxylation reaction involvingphosphoenol-pyruvate carboxylase, is the primary carboxylation reaction.The first stable carbon compound is a 4-carbon acid that is subsequentlydecarboxylated. The CO₂ thus released is refixed by the C₃ cycle.Examples of C₄ plants are tropical grasses, corn, and sugar cane.

Both C₄ and C₃ plants exhibit a range of ¹³C/¹²C isotopic ratios, buttypical values are about −7 to about −13 per mil for C₄ plants and about−19 to about −27 per mil for C₃ plants (see, e.g., Stuiver et al.,Radiocarbon, 19: 355 (1977)). Coal and petroleum fall generally in thislatter range. The ¹³C measurement scale was originally defined by a zeroset by Pee Dee Belemnite (PDB) limestone, where values are given inparts per thousand deviations from this material. The “δ¹³C” values areexpressed in parts per thousand (per mil), abbreviated,

, and are calculated as follows:

δ¹³ C(

)=[(¹³ C/ ¹² C)_(sample)−(¹³ C/ ¹² C)_(standard)]/(¹³ C/ ¹²C)_(standard)×1000

Since the PDB reference material (RM) has been exhausted, a series ofalternative RMs have been developed in cooperation with the IAEA, USGS,NIST, and other selected international isotope laboratories. Notationsfor the per mil deviations from PDB is δ¹³C. Measurements are made onCO₂ by high precision stable ratio mass spectrometry (IRMS) on molecularions of masses 44, 45, and 46.

The branched fatty alcohol and derivative compositions as well as thesurfactants or cleaning compositions described herein includebioproducts produced by any of the methods described herein. Thesurfactants and cleaning compositions can have a δ¹³C of about −28 orgreater, about −27 or greater, −20 or greater, −18 or greater, −15.4 orgreater, −15 or greater, −13 or greater, −10 or greater, or −8 orgreater. A surfactant or cleaning composition so produced can have aδ¹³C of about −30 to about −15, about −27 to about −19, about −25 toabout −21, about −15 to about −5, about −15.4 to about −10.9, about−13.92 to about −13.84, about −13 to about −7, or about −13 to about−10. For example it can have a δ¹³C of about −10, −11, −12, or −12.3.

The surfactants or cleaning compositions herein can also bedistinguished from petroleum-derived compounds by comparing the amountof ¹⁴C in each compound. Because ¹⁴C has a nuclear half life of 5730years, petroleum based chemicals containing “older” carbon can bedistinguished from bioproducts which contain “newer” carbon (see, e.g.,Currie, “Source Apportionment of Atmospheric Particles,”Characterization of Environmental Particles, J. Buffle and H. P. vanLeeuwen, Eds., 1 of Vol. I of the IUPAC Environmental AnalyticalChemistry Series (Lewis Publishers, Inc.) (1992), pp. 3-74).

The basic assumption in radiocarbon dating is that the constancy of ¹⁴Cconcentration in the atmosphere leads to the constancy of ¹⁴C in livingorganisms. But because of atmospheric nuclear testing since 1950 and theburning of fossil fuel since 1850, ¹⁴C has acquired a second,geochemical time characteristic. Its concentration in atmospheric CO₂,and hence in the living biosphere, approximately doubled at the peak ofnuclear testing, in the mid-1960s. It has since been gradually returningto the steady-state cosmogenic (atmospheric) baseline isotope rate(¹⁴C/¹²C) of about 1.2×10⁻¹², with an approximate relaxation “half-life”of 7-10 years. (This latter half-life is not to be taken literally;rather, the detailed atmospheric nuclear input/decay function to tracethe variation of atmospheric and biospheric ¹⁴C since the onset of thenuclear age should be used).

It is this latter biospheric ¹⁴C time characteristic that holds out thepromise of annual dating of recent biospheric carbon. ¹⁴C can bemeasured by accelerator mass spectrometry (AMS), with results given inunits of “fraction of modern carbon” (f_(M)). f_(M) is defined byNational Institute of Standards and Technology (NIST) Standard ReferenceMaterials (SRMs) 4990B and 4990C. As used herein, “fraction of moderncarbon” or “f_(M)” has the same meaning as defined by National Instituteof Standards and Technology (NIST) Standard Reference Materials (SRMs)4990B and 4990C, known as oxalic acids standards HOxI and HOxII,respectively. The fundamental definition relates to 0.95 times the¹⁴C/¹²C isotope ratio HOxI (referenced to AD 1950). This is roughlyequivalent to decay-corrected pre-Industrial Revolution wood. For thecurrent living biosphere (plant material), f_(M) is approximately 1.1.

The invention provides surfactants or cleaning compositions, having anf_(M) ¹⁴C of at least about 1. An exemplary surfactant has an f_(M) ¹⁴Cof at least about 1.01, of at least about 1.5, an f_(M) ¹⁴C of about 1to about 1.5, an f_(M) ¹⁴C of about 1.04 to about 1.18, or an f_(M) ¹⁴Cof about 1.111 to about 1.124. Likewise, an exemplary cleaningcomposition has an f_(M) ¹⁴C of at least about 1.01, of at least about1.5, an f_(M) ¹⁴C of about 1 to about 1.5, an f_(M) ¹⁴C of about 1.04 toabout 1.18, or an f_(M) ¹⁴C of about 1.111 to about 1.124.

Another measurement of ¹⁴C is known as the percent of modern carbon,pMC. For an archaeologist or geologist using ¹⁴C dates, AD 1950 equals“zero years old”. This also represents 100 pMC. “Bomb carbon” in theatmosphere reached almost twice the normal level in 1963 at the peak ofthermonuclear weapons testing. Its distribution within the atmospherehas been approximated since its appearance, showing values that aregreater than 100 pMC for plants and animals living since AD 1950. It hasgradually decreased over time with today's value being near 107.5 pMC.This means that a fresh biomass material, such as corn, would give a ¹⁴Csignature near 107.5 pMC. Petroleum based compounds will have a pMCvalue of zero. Combining fossil carbon with present day carbon willresult in a dilution of the present day pMC content. By presuming 107.5pMC represents the ¹⁴C content of present day biomass materials and 0pMC represents the ¹⁴C content of petroleum based products, the measuredpMC value for that material will reflect the proportions of the twocomponent types. For example, a material derived 100% from present daysoybeans would give a radiocarbon signature near 107.5 pMC. If thatmaterial was diluted 50% with petroleum based products, it would give aradiocarbon signature of approximately 54 pMC.

A biologically based carbon content is derived by assigning “100%” equalto 107.5 pMC and “0%” equal to 0 pMC. For example, a sample measuring 99pMC will give an equivalent biologically based carbon content of 93%.This value is referred to as the mean biologically based carbon resultand assumes all the components within the analyzed material originatedeither from present day biological material or petroleum based material.

A surfactant or a cleaning composition comprising branched fattyalcohols and/or derivatives described herein can have a pMC of at leastabout 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100. In otherinstances, such a surfactant or cleaning composition can have a pMC ofbetween about 50 and about 100; between about 60 and about 100; betweenabout 70 and about 100; between about 80 and about 100; between about 85and about 100; between about 87 and about 98; or between about 90 andabout 95. In yet other instances, it can have a pMC of about 90, 91, 92,93, 94, or 94.2.

Accordingly the present invention is drawn to a branched fatty alcoholor a derivative thereof produced by an engineered microbial host cell.The engineered microbial host cell expresses: (a) a first gene encodinga first polypeptide having at least about 85% sequence identity to theamino acid sequence of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and15, or of a variant thereof; and (b) a second gene encoding a secondpolypeptide having at least about 85% sequence identity to the aminoacid sequence of any one or SEQ ID NOs:24, 26, 28, 30, 32, 34, 36, and38, or of a variant thereof, and is cultured in the presence of one ormore biological substrates of the first and second polypeptides. In someembodiments, the microbial host cell is engineered to express a thirdgene encoding a third polypeptide comprising an amino acid sequencehaving at least an about 85% sequence identity to the amino acidsequence of any one of SEQ ID NOs:47, 49, 51, 53, 55, 57, 59, and 61, orof a variant thereof. In some embodiments, the microbial host cell isengineered to express a fourth gene encoding a fourth polypeptidecomprising an amino acid sequence having at least an about 85% sequenceidentity to the amino acid sequence of any one of SEQ ID NO:69, 71, 73,75, 77, 79, 81, and 83, or of a variant thereof. In any of the aboveembodiments, the microbial host cell is engineered to express abeta-ketoacyl ACP synthase gene in the host cell, wherein thebeta-ketoacyl ACP gene encodes a polypeptide comprising an amino acidsequence having at least about 85% sequence identity to the amino acidsequence of any one of SEQ ID NOs:90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, and 120, or of a variant thereof. Thebeta-ketoacyl ACP synthase is, for example, a FabH that has specificityfor branched-chain acyl-CoA substrates. In any of the embodiments above,the microbial host cell is engineered to express a fatty aldehydebiosynthesis polypeptide, or a variant thereof. In any of theembodiments above, the microbial host cell is engineered to express anacyl-ACP reductase polypeptide or a variant thereof, to modify theexpression of a gene encoding a fatty acid synthase, which compriseexpressing a gene encoding a thioesterase in the microbial host cell, toexpress a gene encoding an alcohol dehydrogenase or a variant thereof,and/or to express an attenuated level of a fatty acid degradation enzymerelative to a wild type host cell. The fatty acid degradation enzyme is,for example, an acyl-CoA synthase.

Branched Fatty Alcohol Derivatives

A derivative of the branched fatty alcohol produced in accordance to themethods described herein can be produced by converting the isolatedbranched fatty alcohol into a branched fatty alcohol derivative thereof.The branched fatty alcohol derivative can be any suitable branched fattyalcohol derivative selected from, for example, a branched fatty ethersulfate, a branched fatty phosphate ester, analkylbenzyldimethyl-ammonium chloride, a branched fatty amine oxide, abranched fatty alcohol sulfate, a branched alkyl polyglucoside, abranched alkyl glyceryl ether sulfonate, and a branched ethoxylatedfatty alcohol. Typically, the branched fatty alcohol derivativecomprises an alkyl group that is about 6 to about 26 carbons in length.Preferably, the branched fatty alcohol comprises an alkyl group that isabout 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbons in length.In certain embodiments, the alkyl group comprises one or more points ofbranching. In this regard, the number of carbons in the alkyl grouprefers to the hydrocarbon group derived from the branched fatty alcohol,and not to any carbon atoms added in the preparation of the branchedfatty alcohol derivative, such as polyethoxy groups and the like.

As used herein, the term “fatty ether sulfate” is the same as “alkylether sulfate” wherein the alkyl residue is a fatty residue, and denotesa compound of the structure: RO(CH₂CH₂O)_(n)—SO₃H, wherein R is a C₆-C₂₆alkyl group as defined herein, and n is an integer of 1 to about 50.Fatty ether sulfate can also refer to the salt denoted byRO(CH₂CH₂O)_(n)SO₃X, where n and R are as defined above and X is acation. An exemplary fatty ether sulfate salt is a sodium salt, forexample, RO(CH₂CH₂O)_(n)SO₃Na. In an exemplary embodiment, the R groupcomprises one or more points of branching.

As used herein, the term “fatty alcohol sulfate” denotes a compound ofthe structure: ROSO₃H wherein R is a C₈-C₂₆ alkyl group. Fatty alcoholsulfate can also refer to the salt of the above structure, denoted byROSO₃X where R is as defined above and X is a cation. An exemplary fattyalcohol sulfate salt is a sodium salt, for example, ROSO₃Na. In anexemplary embodiment, the R group comprises one or more points ofbranching.

As used herein, the term “fatty phosphate ester” is the same as “alkylphosphate ester” wherein the alkyl residue is a fatty residue, anddenotes a compound of the structure:

ROP(O)(OH)₂

As used herein, alkylbenzyldimethylammonium chlorides have thestructure:

wherein R is a C₈-C₂₆ alkyl group as defined herein. For example, thealkyl group of R comprises one or more points of branching.

As used herein, the term “fatty amine oxide” is the same as “alkyl amineoxide” wherein the alkyl residue is a fatty residue as defined herein,and denotes a compound of the structure:

wherein R is a C₈-C₂₆ alkyl group as defined herein and wherein R¹ andR² are C₁-C₂₆ alkyl groups, preferably C₁-C₆ alkyl groups. Preferablythe alkyl groups of R, R¹ and R² each independently comprises one ormore points of branching.

Alkyl polyglucosides have the structure: RO(C_(n)H_(2n)O)_(t)Z_(x)wherein R is a C₈-C₂₆ alkyl group, preferably comprising one or morepoints of branching, Z is a glucose residue, n is 2 or 3, t is from 0 to10, and x is from about 1 to 10, preferably from about 1.5 to 4.

Alkyl glyceryl ether sulfonates have the structure:

wherein R is a C₈-C₂₆ alkyl group as defined herein, preferablycomprising one or more points of branching, and n is an integer from 1to 4, for example, 1, 2, 3, or 4.

As used herein, the term “fatty alcohol alkoxylate” is the same as“alkoxylated fatty alcohol” and denotes a compound of the structure:RO(CH₂CH₂)_(n)OH wherein R is a C₈-C₂₆ alkyl group as defined herein andn is an integer from 1 to about 50. Preferably R comprises one or morepoints of branching.

The branched fatty alcohol derivatives can be produced by any suitablemethod, many of which are known in the art. See, e.g., “Handbook onSoaps, Detergents, and Acid Slurry,” 2^(nd) ed., NIIR Board, AsiaPacific Business Press, Inc., Delhi, India.

In one embodiment, the branched fatty alcohol derivative is anethoxylated branched fatty alcohol, which is also known in the art as abranched fatty alcohol ethoxylate, and has a structure as describedherein. Preferably, the ethoxylated branched fatty alcohol contains fromabout 1 to about 50 moles of ethylene oxide per mole of branched fattyalcohol.

Surfactants or Detersive Surfactants

A surfactant composition of the present invention can comprise about0.001 wt. % to about 100 wt. % of microbially produced branched fattyalcohols and/or derivatives thereof. Preferably, a surfactantcomposition is a blend of a microbially produced branched fatty alcoholand/or derivative in combination with one or more other surfactantsand/or surfactant systems that have been derived from similar (e.g.,microbially derived) or different sources (e.g., synthetic,petroleum-derived). Those other surfactants and/or surfactant systemscan confer additional desirable properties. In some embodiments, the oneor more other surfactants and/or surfactant systems that are blendedwith the microbially produced branched fatty alcohols and/or derivativescan comprise linear or branched fatty alcohol derivatives, or they canbe other types of surfactants such as, cationic surfactants, anionicsurfactants and/or amphoteric/zwitterionic surfactants. These othersurfactants and/or surfactants systems are collectively referred to as“co-surfactants” herein. For example, a surfactant composition of theinvention can be a blend of a microbially produced branched fattyalcohol and/or derivative composition prepared in accordance with thedisclosure herein, and a cationic surfactant derived from apetrochemical source, and the resulting surfactant composition only hasgood cleaning properties but also contributes certain disinfectingand/sanitizing benefits.

The cleaning composition of the invention can comprise, in addition tothe microbially produced branched fatty alcohols and/or derivatives, orthe surfactants comprising such branched materials and/or derivatives,co-surfactants selected from nonionic surfactants, anionic surfactants,cationic surfactants, ampholytic surfactants, squitterionic surfactants,semi-polar nonionric surfactants, and mixtures thereof. When present,the total amount of surfactants, including the microbially producedbranched fatty alcohols and/or derivatives thereof, and theco-surfactants, is typically present at a level of about 0.1 wt. % orhigher (e.g., about 1.0 wt. % or higher, about 10 wt. % or higher, about25 wt. % or higher, about 50 wt. % or higher, about 70 wt. % or higher).For example, the total amount of surfactant in a cleaning compositioncan vary from about 0.1 wt. % to about 80 wt. % (e.g., from about 0.1wt. % to about 40 wt. %, from about 0.1 wt % to about 12 wt. %, fromabout 1.0 wt. % to about 50 wt. %, or from about 5 wt. % to about 40 wt.%).

Various known surfactants can be suitable co-surfactants. In someembodiments, the co-surfactant can comprise an anionic surfactant. Incertain embodiments, the amount of one or more anionic surfactants inthe cleaning composition can be, for example, about 1 wt. % or more(e.g., about 5 wt. % or more, about 10 wt. % or more, about 20 wt. % ormore, about 30 wt. % or more, about 40 wt. % or more). For example, theamount of one or more anionic surfactants in the cleaning compositioncan vary from about 1 wt. % to about 40 wt. %. Suitable anionicsurfactants include, for example, linear alkylbenzenesulfonate,alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcoholethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methylesters, alkyl- or alkenylsuccinic acid or soap. In some embodiments, ananonic surfactant is, for example, a C₁₀-C₁₈ alkyl akoxy ester(AE_(x)S), wherein x is from 1-30. Other suitable anionic surfactantscan be found in International Publication WO98/39403, Surface ActiveAgents and Detergetns (Vol. 1, & II, by Schwartz, Perry and Berch), andU.S. Pat. Nos. 3,929,678, 6,020,303, 6,060,443, 6,008,181, InternationalPublications WO 99/05243, WO 99/05242 and WO 99/05244, the relevantdisclosures of which are incorporated herein by reference.

In another embodiment, the co-surfctant can comprise a cationicsurfactant. Suitable cationic surfactants include, for example, thosehaving long-chain hydrocarbyl groups. Examples include ammoniumsurfactants such as alkyltrimethylammonium halodenides, and surfactantshaving the formula [R²(OR³)y][R⁴(OR³)y]₂R⁵N+X⁻, wherein R² is an alkylor alkyl benzyl group having from about 8 to about 18 carbon atoms inthe alkyl chain, each R³ is independently selected from the groupconsisting of —CH₂CH₂—, CH₂CH(CH₃)—, CH₂(CH(CH₂OH))—, CH₂CH₂CH₂—, andmixtures thereof; each R⁴ is selected from the group consisting of C₁-C₄alkyl, C₁-C₄ hydroxyalkyl, benzyl ring structures formed by joining thetwo R⁴ groups, —CH₂CHOH—CHOHCOR⁶CHOHCH₂OH wherein R⁶ is any hexose orhexose polymer having a molecular weight less than about 1,000, andhydrogen, when y is not 0; R⁵ is the same as R⁴ or is an alkyl chainwherein the total number of carbon atoms of R² plus R⁵ is not more thanabout 18; each y is from 0 to about 10 and the sum of the y values isfrom 0 to about 15; and X is any compatible anion.

Certain quaternary ammonium surfactant may also be suitable as cationicco-surfactants, and examples of those are described in InternationalPublication WO 98/39403. Examples of suitable quaternary ammoniumcompounds include coconut trimethyl ammonium chloride or bromide;coconut methyl dihydroxyethyl ammonium chloride or bromide; decyltriethyl ammonium chloride; decyl di methyl hydroxyethyl ammoniumchloride or bromide; C₁₂₋₁₅ dimethyl hydroxyethyl ammonium chloride orbromide; coconut dimethyl hydroxyethyl ammonium chloride or bromide;myristyl trimethyl ammonium methyl sulphate; lauryl dimethyl benzylammonium chloride or bromide; lauryl di methyl (ethenoxy) 4 ammoniumchloride or bromide. Other cationic surfactants have been described inU.S. Pat. Nos. 4,228,044, 4,228,042, 4,239,660 4,260,529 6,136,769,6,004,922, 6,022,844, and 6,221,825, International Publications WO98/35002, WO 98/35003, WO 98/35004, WO 98/35005, WO 98/35006, and WO00/47708, as well as European Patent Application EP 000,224. Whenincluded herein, the surfactant/detergent and the cleaning/treatmentcompositions of the present invention can comprise, for example, fromabout 0.2 wt. % to about 25 wt. %, preferably from about 1 wt. % toabout 8 wt. % by weight of cationic surfactants.

In certain embodiments, co-surfactants can comprise nonionicsurfactants. Polyethylene, polypropylene, and polybutylene oxidecondensates of alkyl phenols are suitable, with the polyethylene oxidecondensates being preferred. They include the condensation products ofalkyl phenols having an alkyl group having about 6 to about 14 carbonatoms, preferably from about 8 to about 14 carbon atoms, in either astraight-chain or branched-chain configuration, with alkylene oxide. Inparticular embodiments, the ethylene oxide is present in an amount offrom about 2 to about 25 moles (e.g., from about 3 to about 15 moles) ofethylene oxide per mole of alkyl phenol. Commercially available nonionicsurfactants of this type include Igepal™ C0-630 (The GAF Corp.), Triton™X-45, X-114, X-100 and X-102 (Dow Chemicals). These surfactants arecommonly referred to as alkylphenol alkoxylates (e.g., alkyl phenolethoxylates).

Moreover, condensation products of primary and secondary aliphaticalcohols with from about 1 to about 25 moles of ethylene oxide aresuitable nonionic co-surfactants. The alkyl chain of the aliphaticalcohol can be straight or branched, primary or secondary, and generallycan contain about 8 to about 22 (e.g., about 8 to about 20, or about 10to about 18) carbon atoms with about 2 to about 10 moles (e.g., about 2to about 5 moles) of ethylene oxide per mole of alcohol present in thecondensation products. Examples of commercially available nonionicsurfactants of this type include Tergitol™ 15-S-9, Tergitol™ 24-L-6 NMW(Union Carbide); Neodol™ 45-9, Neodol™ 23-3, Neodol™ 45-7, Neodol™ 45-5(Shell Chemical), Kyro™ EOB (Procter & Gamble), and Genapol LA 030 or050 (Hoechst).

Further examples of nonionic co-surfactants include C₁₂-C₁₈ alkylethoxylates (e.g., NEODOL® nonionic surfactants (Shell)), C₆-C₁₂ alkylphenol alkoxylates wherein the alkoxylate units are a mixture ofethyleneoxy and propyleneoxy units, C₁₂-C₁₈ alcohol and C₆-C₁₂ alkylphenol condensates with ethylene oxide/propylene oxide block alkylpolyamine ethoxylates (e.g., PLURONIC® (BASF)), C₁₄-C₂₂ mid-chainbranched alcohols as described in U.S. Pat. No. 6,150,322, C₁₄-C₂₂mid-chain branched alkyl alkoxylates, BAE_(x), wherein x is from 1-30,as described in U.S. Pat. Nos. 6,153,577, 6,020,303 and 6,093,856,alkylpolysaccharides as described in U.S. Pat. No. 4,565,647,alkylpolyglycosides as described in U.S. Pat. No. 4,483,780 and U.S.Pat. No. 4,483,779, polyhydroxy detergent acid amides as described inU.S. Pat. No. 5,332,528, or ether capped poly(oxyalkylated) alcoholsurfactants as described in U.S. Pat. No. 6,482,994 and InternationalPublication WO 01/42408.

Semi-polar nonionic surfactants can also be suitable. They include,e.g., water-soluble amine oxides containing 1 alkyl moiety of from about10 to about 18 carbon atoms and 2 moieties selected from alkyl orhydroxyalkyl moieties containing about 1 to about 3 carbon atoms,water-soluble phosphine oxides containing 1 alkyl moiety of about 10 toabout 18 carbon atoms and 2 moieties selected from alkyl or hydroxyalkylmoieties containing about 1 to about 3 carbon atoms; and water-solublesulfoxides containing 1 alkyl moiety of about 10 to about 18 carbonatoms and a moiety selected from alkyl or hydroxyalkyl moieties of about1 to about 3 carbon atoms. Semi-polar nonionic surfactants have beendescribed in, e.g., International Publication WO 01/32816, U.S. Pat.Nos. 4,681,704 and 4,133,779.

Moreover, alkylpolysaccharides, such as those described in U.S. Pat. No.4,565,647, having a hydrophobic group containing about 6 to about 30carbon atoms (e.g., from about 10 to about 16 carbon atoms) and apolysaccharide, can also be suitable semi-polar noniornicco-surfactants. Others have been described in, for example,International Publication WO 98/39403. When included herein, thecleaning/treatment compositions of the present invention can comprise,for example, about 0.2 wt. % or more (e.g., about 1 wt. % or more, about5 wt. % or more, or about 8 wt. % or more) of such semi-polar nonionicsurfactants. For example, the cleaning compositions of the invention cancomprise about 0.2 wt. % to about 15 wt. % (e.g., about 1 wt. % to about10 wt. %) of semi-polar nonionic surfactants.

In certain embodiments, the co-surfactants comprises ampholyticsurfactants. Ampholytic surfactants can be broadly described asaliphatic derivatives of secondary or tertiary amines, or aliphaticderivatives of heterocyclic secondary and tertiary amines in which thealiphatic radical can be straight- or branched-chain. One of thealiphatic substituents can contain at least about 8 carbon atoms (e.g.,from about 8 to about 18 carbon atoms), and at least another contains ananionic water-solubilizing group, e.g. carboxy, sulfonate, or sulfate.Ampholytica surfactants have been described in, for example, U.S. Pat.No. 3,929,678. When included therein, a cleaning composition of theinvention can comprise, for example, about 0.2 wt. % to about 15 wt. %(e.g., about 1 wt. % to about 10 wt. %) of ampholytic surfactants.

In certain other embodiments, especially in personal carecleaning/treatment compositions, zwitterionic surfactants are includedas co-surfactants. These surfactants can be broadly described asderivatives of secondary and tertiary amines, derivatives ofheterocyclic secondary and tertiary amines, or derivatives of quaternaryammonium, quaternary phosphonium or tertiary sulfonium compounds.Zwitterionic surfactants have been described in, for example, U.S. Pat.No. 3,929,678. When included therein, a surfactant/detergent orcleaning/treatment composition of the invention can comprise, forexample, about 0.2 wt. % to about 15 wt. % (e.g., about 1 wt. % to about10 wt. %) of zwitterionic surfactants.

In further embodiments, primary or tertiary amines can be included asco-surfactants. Suitable primary amines include amines according to theformula R¹NH₂ wherein R¹ is a C₆-C₁₂, preferably C₆-C₁₀, alkyl chain; orR₄X(CH₂)n, wherein X is —O—, —C(O)NH— or —NH—, R⁴ is a C₆-C₁₂ alkylchain, n is between 1 to 5 (e.g., 3). The alkyl chain of R¹ can bestraight or branched, and can be interrupted with up to 12, butpreferably less than 5 ethylene oxide moieties. Preferred amines includen-alkyl amines, selected from, for example, 1-hexylamine, 1-octylamine,1-decylamine and laurylamine, C₈-C₁₀ oxypropylamine,octyloxypropylamine, 2-ethylhexyl-oxypropylamine, lauryl amidopropylamine or amido propylamine. Suitable tertiary amines include thosehaving the formula R¹R²R³N wherein R¹ and R² are C₁-C₈ alkylchains, R³is either a C₆-C₁₂, preferably C₆-C₁₀, alkyl chain, or R³ is R⁴X(CH₂)n,whereby X is —O—, —C(O)NH— or —NH—, R⁴ is a C₄-C₁₂, n is between 1 and 5(e.g., 2, 3, or 4), R⁵ is H or C₁-C₂ alkyl, and x is between 1 and 6. R³and R⁴ may be linear or branched. The alkyl chain of R³ can beinterrupted with up to 12, but preferably less than 5, ethylene oxidemoieties. Preferred tertiary amines include, for example, 1-hexylamine,1-octylamine, 1-decylamine, 1-dodecylamine, n-dodecyldimethylamine,bishydroxyethylcoconutalkylamine, oleylamine(7)ethoxylated, lauryl amidopropylamine, and cocoamido propylamine. Other useful detersivesurfactants have been described in the prior art, for example, in U.S.Pat. Nos. 3,664,961, 3,919,678, 4,222,905, and 4,239,659.

In some embodiments, the detergent/cleaning composition of the inventioncomprises greater than about 5 wt. % anionic surfactant and/or less thanabout 25 wt. % nonionic surfactant. For example, the compositioncomprises greater than about 10 wt. % anionic surfactants. In anotherexample, the composition comprises less than 15%, more preferably, lessthan 12% nonionic surfactants.

The total amount of surfactants included in a cleaning composition ofthe invention is typically about 0.1 wt. % or more (e.g., about 1 wt. %or more, about 10 wt. % or more, about 25 wt. % or more, about 50 wt. %or more, about 60 wt. % or more, about 70 wt. % or more). An exemplarycleaning composition of the invention comprises about 0.1 wt. % to about80 wt. % total surfactants (e.g., about 1 wt. % to about 50 wt. %, about10 wt. % to about 40 wt. %, about 20 wt. % to about 35 wt. %) of totalsurfactants, including the microbially produced branched fatty alcoholsand/or derivatives thereof and co-surfactants.

One criteria based on which to the type(s) and amount(s) of surfactantsto be included in cleaning compositions can be determined iscompatibility with the enzyme components present in the cleaningcompositions. For example, in liquid or gel compositions, the cleaningcomposition (including all the surfactants, which are, for example,pre-formulated into a surfactant package) is prepared such that itpromotes, or at least does not degrade, the stability of any enzyme inthe cleaning composition.

A surfactant composition of the present invention, or a surfactantpackage, which can be formulated and subsequently included in a cleaningcomposition, can be in any form, for example, a liquid; a solid such asa powder, granules, agglomerate, paste, tablet, pouches, bar; a gel; anemulsion; or in a suitable form to be delivered in dual-compartmentcontainers. The composition can also be formulated into a spray or foamdetergent, premoistened wipes (e.g., the cleaning composition incombination with a nonwoven material as described, for example, in U.S.Pat. No. 6,121,165), dry wipes (e.g., a cleaning composition incombination with a nonwoven material, activated with water by aconsumer, as described, for example, in U.S. Pat. No. 5,980,931), andother homogeneous or multiphase consumer cleaning product forms.

Cleaning Compositions

Surfactant compositions comprising branched fatty alcohols and/orderivatives thereof, e.g., sulfate, alkoxyalated or alkoxylated sulfatederivatives, are particularly suitable as soil detachment-promotingingredients of laundry detergents, dishwashing liquids and powders, andvarious other cleaning compositions. They exhibit good dissolving powerespecially when faced with greasy soils, and it is particularadvantageous that they display the outstanding soil-detaching power evenat low washing temperatures.

The branched fatty alcohol/derivative compositions of the presentinvention can be included or blended into a surfactant package asdescribed above, which comprises about 0.0001 wt. % to about 100 wt. %of one or more branched fatty alcohols and/or derivatives produced by agenetically engineered host cell or microbe. That surfactant package canthen be blended into a cleaning composition to impart detergency andcleaning power to the cleaning composition. In alternative embodiments,the branched fatty alcohols and/or derivatives thereof produced by thehost cell or mibrobe can be blended into a cleaning compositiondirectly, in an amount of about 0.001 wt. % or more (e.g., about 0.001wt. % or more, about 0.1 wt. % or more, about 1 wt. % or more, about 10wt. % or more, about 20 wt. % or more, or about 40 wt. % or more) basedon the total weight of the cleaning composition. For example, thebranched fatty alcohols and/or derivatives thereof can be blended into acomposition in an amount of about 0.001 wt. % to about 50 wt. % (e.g.,about 0.01 wt. % to about 45 wt. %, about 0.1 wt. % to about 40 wt. %,about 1 wt. % to about 35 wt. %). Accordingly, a cleaning composition ofthe present invention, in either a solid form (e.g., a tablet, granule,powder, or compact), or a liquid form (e.g., a fluid, gel, paste,emulsion, or concentrate) can comprise about 0.001 wt. % to about 50 wt.% of microbially produced branched fatty alcohols and/or derivativesthereof. For example, a cleaning composition of the invention cancomprise about 0.5 wt. % to about 44 wt. % of microbially producedbranched fatty alcohols and/or derivatives thereof. Preferably, thecleaning composition comprises about 1 wt. % to about 30 wt. % ofmicrobially produced branched fatty alcohols and/or derivatives.

Alternatively, a cleaning composition of the present invention cancomprise about 0.001 wt. % to about 80 wt. % of a surfactant packageformulated to comprise about 0.001 wt. % to about 100 wt. % ofmicrobially produced branched fatty alcohols and/or derivatives. Forexample, a cleaning composition of the present invention can compriseabout 0.1 wt. % to about 50 wt. % of such a surfactant package. Thesurfactant package can comprise other surfactants (i.e.,co-surfactants), which can include surfactants derived from similar(e.g., microbially-produced surfactant) or different sources (e.g.,petroleum-derived surfactants). In a particular embodiment, however, thesurfactant package can comprise mostly or exclusively of branched fattyalcohols and/or derivatives produced by a host cell or a microbe asdescribed herein.

Industrial Cleaning Compositions, Household Cleaning Compositions &Personal Care Cleaning Compositions

In certain embodiments, the cleaning composition of the presentinvention is a liquid or solid laundry detergent composition. In someembodiments, the cleaning composition is a hard surface cleaningcomposition, wherein the hard surface cleaning composition preferablyimpregnates a nonwoven substrate. As used herein, “impregnate” meansthat the hard surface cleaning composition is placed in contact with anonwoven substrate such that at least a portion of the nonwovensubstrate is penetrated by the hard surface cleaning composition. Forexample, the hard surface cleaning composition preferably saturates thenonwoven substrate. In other embodiments, the cleaning composition ofthe present invention is a car care composition, which is useful forcleaning various surfaces such as hard wood, tile, ceramic, plastic,leather, metal, and/or glass. In some embodiments, the cleaningcomposition is a dish-washing composition, such as, for example, aliquid hand dishwashing composition, a solid automatic dishwashingcomposition, a liquid automatic dishwashing composition, and a tab/unitdose form automatic dishwashing composition.

In further embodiments, the cleaning composition can be used inindustrial environments for cleaning of various equipment, machinery,and for use in oil drilling operations. For example, the cleaningcomposition of the present invention can be particularly suited inenvironments wherein it comes into contact with free hardness and incompositions that require hardness tolerant surfactant systems, such aswhen used to aid oil drilling.

In some embodiments, the cleaning composition of the invention can beformulated into personal or pet care compositions such as shampoos, bodywashs, or liquid or solid soaps.

Common cleaning adjuncts applicable to most cleaning compositions,including, household cleaning compositions, and personal carecompositions and the like, include builders, enzymes, polymers, sudsboosters, suds suppressors (antifoam), dyes, fillers, germicides,hydrotropes, anti-oxidants, perfumes, pro-perfumes, enzyme stabilizingagents, pigments, and the like. In some embodiments, the cleaningcomposition is a liquid cleaning composition, wherein the compositioncomprises one or more selected from solvents, chealating agents,dispersants, and water. In other embodiments, the cleaning compositionis a solid, wherein the composition further comprises, for example, aninorganic filler salt. Inorganic filler salts are conventionalingredients of solid cleaning compositions, present in substantialamounts, varying from, for example, about 10 wt. % to about 35 wt. %.Suitable filler salts include, for example, alkali and alkaline-earthmetal salts of sulfates and chlorides. An exemplary filler salt issodium sulfate.

Household cleaning compositions, including, e.g., laundry detergents andhousehold surface cleaners typically comprise certain additional, insome embodiments, more specialized, ingredients or cleaning adjunctsselected from one or more of: bleaches, bleach activators, catalyticmaterials, suds boosters, suds suppressors (antifoams), diverse activeingredients or specialized materials such as dispersant polymers (e.g.,dispersant polymers sold by BASF or Dow Chemicals), silvercare,anti-tarnish and/or anti-corrosion agents, dyes, germicides, alkalinitysources, hydrotropes, anti-oxidants, enzyme stabilizing agents,pro-perfumes, perfumes, solubilizing agents, carriers, processing aids,pigments, and, for liquid formulations, solvents, chelating agents, dyetransfer inhibiting agents, dispersants, brighteners, dyes, structureelasticizing agents, fabric softeners, anti-abrasion agents,hydrotropes, processing aids, and other fabric care agents. The cleaningadjuncts particularly useful for household cleaning compositions and thelevels of use have been described in, e.g., U.S. Pat. Nos. 5,576,282,6,306,812 and 6,326,348. A comprehensive list of suitable lanudry orother household cleaning adjuncts is, e.g., in International PublicationWO 99/05245.

Personal/pet or beauty care cleaning compositions including, e.g.,shampoos, facial cleansers, hand sanitizers, bodywash, and the like, canalso comprise, in some embodiments, other more specialized adjuncts,inlcuding, for example, conditioning agents such as vitamines, silicone,silicone emulsion stabilizing components, cationic cellulose or polymerssuch as Guar polymers, anti-dandruff agents, antibacterial agents,dispersed gel network phase, suspending agents, viscosity modifiers,dyes, non-volatile solvens or diluents (water soluble or insoluble),foam boosters, pediculocides, pH adjusting agnets, perfumes,preservatives, chelants, proteins, skin active agents, sunscreens, UVabsorbers, and minerals, herbal/fruit/food extracts, sphigolipidsderivatives or synthetic derivatives and clay.

Common Adjuncts

(1) Enzymes

Various known detersive enzymes can be blended into a cleaningcomposition of the present invention. Suitable enzymes include, e.g.,proteases, amylases, lipases, cellulases, pectinases, mannases,arabinases, galactanases, xylanases, oxidases (e.g., laccases),peroxidases, and/or mixtures thereof. They can provide enhanced cleaningperformance and/or fabric care benefits. In general, just as theselection of the type and amount of surfactants to be formulated into acleaning composition should take account of the enzymes therein, thetypes of enzyme chosen to be included in the composition should takeaccount of the other components in the composition (including thesurfactants). Considerations may include, e.g., the pH-optimum of theoverall composition, the presence of absence of enzyme stabilizationagents, etc. The enzymes should be present in the cleaning compositionsin effective amounts.

Suitable proteases include those of animal, vegetable or microbialorigin. Microbial origin is preferred. Chemically modified or engineeredmutants (e.g., those desecribed in International Publications WO92/19729, 98/20115, 98/20116, and 98/34946) can also be included.Suitable proteases can be a serine protease or a metallo protease,preferably an alkaline microbial protease or a trypsin-like protease.Examples of alkaline proteases are subtilisins, especially those derivedfrom Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin309, subtilisin 147 and subtilisin 168 (as described in InternationalPublications WO 89/06279 and WO 05/103244). Other suitable serineproteases include those from Micrococcineae sp. and those fromCellulonas sp. and variants thereof as, e.g., described in InternationalPublication WO05/052146. Examples of trypsin-like proteases includetrypsin (e.g. of porcine or bovine origin) and Fusarium proteases suchas those described in International Publications WO 89/06270 and WO94/25583. Many proteases are commercially available, including, e.g.,Alcalase™, Savinase™, Primase™, Duralase™, Esperase™ Coronase™,Polarzyme™, Kannase™ (Novozymes A/S), Maxatase™, Maxacal™ Maxapem™,Properase™, Purafect™, Purafect Prime™, Purafect OxP®, FNA, FN2, FN3,and FN4 (Genencor Int'l Inc.).

Suitable lipases include those of bacterial or fungal origin. Forexample, suitable lipases can be derived from yeast, from genera such asCandida, Kluyvermyces, pichia, Saccharomyces, Schizosaccharomyces, orYarrowia, or derived from a filamentous fungus, such as Acremonium,Aspergillus, Aureobasidum, Cryptococcus, Filobasidium, Fusarium,Humicolar, Magnaporthe, Mucor, Myceliophthora, Neocallimasix,Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum,Talaromyces, thermoascus, Thielavia, Tolypocladium, Thermomyces orTrichoderma. Many chemically modified lipases are also suitable,including, for example, those from Humicola, (e.g., a modified lipasefrom H. lanuginosa as described in EP 258 068 and 305 216, a modifiedlipase from H. insolens as described in International Publication WO96/13580), those from Pseudomonas (e.g., a modified lipase from P.alcaligenes, or from P. pseudoalcaligenes as described in EP 218 272, amodified lipase from P. cepacia as described in EP 331 376, a modifiedlipase from P. stutzeri as described in GB 1,372,034, a modified lipasefrom P. fluoresces or Pseudomonas sp. strain SD 705, as described inInternational Publications WO 95/06720 and WO96/27002, a modified lipasefrom P. wisconsinensis as described in International Publication WO96/12012), those from Bacillus (e.g. a modified lipase from B. subtilisas described in Dartois et al. Biochemica et Biophysica Acta, 1131,253-360 (1993)), a modified lipase from B. stearothermophilus asdescribed in JP Application 64/744992, a modified lipase from B. pumilusas described in International Publication WO 91/16422. Other examplesare lipase variants include those described in InternationalPublications WO 92/05249, WO 94/01541, WO 95/35381, WO 96/00292, WO95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079, WO97/07202, and EP 407 225 and 260 105.

A number of lipase enzymes, which can be included in a cleaningcomposition of the invention, are commercially available. They includeLipolase™, Lipolase™ Ultra and Lipex™ (Novozymes A/S). Suitable amylases(α and/or β) include those of bacterial or fungal origin. Chemicallymodified or engineered mutant amylases can also be suitably included ina cleaning composition of the invention. Amylases include, for example,α-amylases obtained from Bacillus (for example, from a special strain ofB. licheniformis as described GB Patent 1,296,839). Various mutantamylases, which can be suitably included in a cleaning composition, havebeen described in International Publications WO 94/02597, WO 94/18314,WO 96/23873, and WO 97/43424. A number of amylases, which can beincluded in a cleaning composition of the present invention, arecommercially available. They include Duramyl™ Termamyl™, Stainzyme™,Stainzyme Ultra™, Stainzyme Plus™, Fungamyl™ and BAN™ (Novozymes A/S),Rapidase™ and Purastar™ (Genencor Int'l Inc.).

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or engineered mutant cellulases can also be suitablyincluded in a cleaning composition of the invention. Cellulases include,for example, those obtained from the genera Bacillus, Pseudomonas,Humicola (e.g., from Humicola insolens), Fusarium (e.g., from Fusariumoxysporum), Thielavia, Acremonium, Myceliophthora, as described in U.S.Pat. Nos. 4,435,307, 5,648,263, 5,691,178, 5,776,757, and InternationalPublication WO 89/09259. Especially suitable cellulases are the alkalineor neutral cellulases that impart color care benefits. Examples of suchcellulases include those described in EP 0 495 257, 0 531 372, andInternational Publications WO 96/11262, WO 96/29397, and WO 98/08940. Anumber of cellulases, especially those that provide added color carebenefits, are commercially available, which can be included in acleaning composition of the invention, especially in, for example, alaundry detergent composition. Commercially available cellulasesinclude, e.g., Renozyme™ Celluclean™, Endolase™, Celluzyme™, andCarezyme™ (Novozymes A/S), Clazinase™ and Puradax HA™ (GenencorInternational Inc.), and KAC-500(B)™ (Kao Corporation).

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or engineered mutantperoxidases/oxidases can also be suitably included in a cleaningcomposition of the invention. Useful peroxidases include, for example,those obtained from the genera Coprinus (e.g., a periosidase from C.cinereus and variants thereof as described in International PublicationsWO 93/24618, WO 95/10602, and WO 98/15257). Commercially availableperoxidases include, for example, Guardzyme™ (Novozymes A/S).

Suitable enzymes described above can be present in a cleaningcomposition of the present invention at levels of about 0.00001 wt. % orhigher (e.g., about 0.01 wt % or higher, about 0.1 wt. % or higher,about 0.5 wt. % or higher, or about 1 wt. % or higher). For example, oneor more such enzymes can be present in a cleaning composition of theinvention in an amount of about 0.00001 wt. % to about 2 wt. % (e.g.,about 0.0001 wt. % to about 1 wt. %, about 0.001 wt. % to about 0.5 wt.%) based on the total weight of the cleaning composition. In certainembodiments, the enzyme(s) can be present or used at very low levels,for example, at about 0.001 wt. % or lower. In alternative embodiments,enzyme(s) can be formulated, for example, into a heavier duty laundrydetergent composition, at about 0.1 wt. % and higher, for example, atabout 0.5 wt. % or higher.

2) Enzyme Stabilizers

In certain embodiments, the cleaning composition of the presentinvention, which comprises one or more enzymes, further comprises one ormore enzyme stabilizers. For example, the enzymes employed in thecleaning composition can be stabilized by the presence of water-solublesources of calcium and/or magnesium ions in the finished compositionsthat provide such ions to the enzymes. Known stabilizing agents include,for example, a polyol such as propylene glycol or a glycerol, a sugar ora sugar alcohol, a lactic acid, a boric acid, a boric acid derivativesuch as an aromatic borate ester, a phenyl boronic acid derivative suchas a 4-formylphenyl boronic acid. The enzyme stabilizers can beincorporated into the cleaning composition according to known methods,such as, for example, those described in International Publications WO92/19709 and WO 92/19708.

3) Builders

Cleaning compositions of the present invention optionally comprise oneor more detergent builders or builder systems. When a builder is used,the subject composition can comprise, e.g., at least about 1 wt. %(e.g., at least about 1 wt. %, at least about 5 wt. %, at least about 10wt. %, at least about 20 wt. %, at least about 30 wt. %, at least about40 wt. %, at least about 50 wt. %, or more) of one or more builders. Forexample, a solid cleaning composition of the present invention cancomprise, e.g., about 1 wt. % to about 60 wt. % (e.g., about 5 wt. % toabout 50 wt. %, about 10 wt. % to about 40 wt. %, about 15 wt. % toabout 30 wt. %) of one or more builders or a builder system. Forexample, a liquid cleaning composition of the present invention cancomprise about 0 wt. % to about 10 wt. % of one or more detergencybuilders.

Various known builder materials can be used, including, e.g.,aluminosilicate materials, silicates, polycarboxylates, alkyl- oralkenyl-succinic acid, and fatty acids, materials such asethylenediamine tetraacetate, diethylene triamine pentamethyleneacetate,metal ion sequestrants such as aminopolyphosphonates, particularlyethylenediamine tetramethylene phosphonic acid and diethylene traminepentamethylenephosphonic acid. Particularly, builder materials such ascalcium sequestrant materials, precipitating materials, calciumion-exchange mateirals, polycarboxylate materials, citrate builder,succinic acid builders, aminocarboxylates, and mixtures thereof arepreferred.

Examples of calcium sequenstrant builder materials include alkali metalpolyphosphates, such as sodium tripolyphosphate and organicsequestrants, and ethylene diamine tetra-acetic acid. Examples ofprecipitating builder materials include sodium orthophosphate and sodiumcarbonate. Examples of calcium ion-exchange builder materials includethe water-insoluble crystalline or amorphous aluminosilicates, of whichzeolites are the best known, e.g., zeolite A, zeolite B (also known aszeolite P), zeolite C, zeolite X, zeolite Y, and also the zeolite P-typeas described in, e.g., EP 0 384 070.

Of particular importance are citrate builders, including, for example,citric acid and soluble salts thereof (particularly sodium salt), arepolycarboxylate builders of particular importance for heavy duty liquiddetergent formulations due to their availability from renewableresources and their biodegradability. Oxydisuccinates are alsoespecially useful in such compositions and combinations. Useful succinicacid builders can also be C₅-C₂₀ alkyl and alkenyl succinic acids andsalts thereof, including laurylsuccinate, myristylsuccinate,palmitylsuccinate, 2-dodecenylsuccinate, 2-pentadecenylsuccinate. withdodecenylsuccinic acid being particularly preferred.

Suitable polycarboxylate builders include, for example, cycliccompounds, particularly alicyclic compounds, such as those described inU.S. Pat. Nos. 3,308,067, 3,723,322, 3,835,163; 3,923,679; 4,102,903,4,120,874, 4,144,226, and 4,158,635.

Ether hydroxypolycarboxylates, copolymers of maleic anhydride withethylene or vinyl methyl ether, 1, 3, 5-trihydroxy benzene-2, 4,6-trisulphonic acid, and carboxymethyl oxysuccinic acid, various alkalimetal, ammonium, and substituted ammonium salts of poly acetic acidssuch as ethylenediamine tetraacetic acid and nitrilotriacetic acid, andpolycarboxylates such as mellitic acid, succinic acid, oxy-disuccinicacid, polymaleic acid, benzene 1,3,5-tricarboxylic acid,carboxymethyloxy-succinic acid, and soluble salts thereof can be used asbuilders. Other nitrogen-containing, phospho-free aminocarboxylates aresometimes used. Specific examples include ethylene diamine disuccinicacid and salts thereof (ethylene diamine disuccinates, EDDS), ethylenediamine tetraacetic acid and salts thereof (ethylene diaminetetraacetates, EDTA), and diethylene triamine penta acetic acid andsalts thereof (diethylene triamine penta acetates, DTPA). In particularembodiments of a liquid composition,3,3-dicarboxy-4-oxa-1,6-hexanedioates and related compounds as describedin U.S. Pat. No. 4,566,984 can be suitable.

4) Chelating Agents

Cleaning compositions of the present invention can optionally compriseone or a mixture of more than one copper, iron and/or manganesechelating agents. When such an agent is used, the subject cleaningcomposition can comprise, for example, about 0.005 wt. % or more (e.g.,about 0.01 wt. % or more, about 1 wt. % or more, about 5 wt. % or more,about 10 wt. % or more) chelating agents. For example, a cleaningcomposition of the invention comprises about 0.005 wt. % to about 15 wt.% (e.g., about 0.01 wt. % to about 12 wt. %, about 0.1 wt. % to about 10wt. %, about 1 wt. % to about 8 wt. %, about 2 wt. % to about 6 wt. %)chelating agents.

Suitable chelating agents include, e.g., amino carboxylates, aminophosphonates, polyfunctionally-substituted aromatic chelating agents, ormixtures, which are capable of removing copper, iron, or manganese ionsfrom washing mixtures by forming soluble chelates.

Amino carboxylates include, for example, ethylenediaminetetracetates,N-hydroxyethylethylenediaminetriacetates, nitrilotriacetates,ethylenediamine tetraproprionates, triethylenetetraamine-hexacetates,diethylenetriamine penta-acetates, and ethanoldiglycines, alkali metal,ammonium, and substituted ammonium salts thereof.

Amino phosphonates are selectively used in cleaning compositions becuasethey increase the amount of total phosphorus. For some applicationswherein the amount of total phosphorus in a cleaning composition islimited, amino phosphonates may not be a suitable chelating agent orshould be used in low amounts. Amino phosphonates include, e.g.,ethylenediamine tetrakis (methylenephosphonates). The amino phosphonatespreferably do not contain alkyl or alkenyl groups with more than about 6carbon atoms.

Suitable polyfunctionally-substiuted aromatic chelating agents have beendescribed in, e.g., U.S. Pat. No. 3,812,044. Exemplarypolyfunctionally-substituted aromatic chelating agents include adihydroxydisulfobenzene, such as a 1,2-dihydroxy-3,5-disulfobenzene.

In some embodiments, biodegradable chelators can be included in acleaning composition of the invention. An exemplary biodegradablechelator is ethylenediamine disuccinate (“EDDS”), especially the [S,S]isomer as described in U.S. Pat. No. 4,704,233.

The compositions herein may also contain water-soluble methyl glycinediacetic acid (MGDA) salts (or acid form) as a chelant or co-builderuseful with, for example, insoluble builders such as zeolites, layeredsilicates and the like.

5) Hydrotropes

Hydrotropes can be optionally included in cleaning compositions of thepresent invention to improve the physical and chemical stability of thecompositions. Suitable hydrotropes include sulfonated hydrotropes, whichinclude, for example, alkyl aryl sulfonates, or alkyl aryl sulfonicacids. Alkyl aryl sulfonates can be sodium, potassium, calcium, orammonium xylene sulfonates; sodium, potassium, calcium, or ammoniumtoluene sulfonates; sodium, potassium, calcium, or ammonium euraenesulfonates; sodium, potassium, calcium, or ammonium substituted orunsubstituted naphthalene sulfonates, and mixtures thereof. Preferredamong these are the sodium salts. Alkyl aryl sulfonic acids can bexylenesulfonic acid, toluenesuifonic acid, cumenesulfonic acid,substituted or unsubstituted naphthalenesulfonic acid, or salts thereof.In certain embodimens, a mixture of xylenesulfonic acid and p-toluenesulfonate can be used.

If present, a cleaning composition of the present invention compriseshydrotropes in an amount of about 0.01 wt. % or more (e.g., about 0.02wt. % or more, about 0.05 wt. % or more, about 0.1 wt. % or more, about1 wt. % or more, about 5 wt. % or more, about 10 wt. % or more, or about15 wt. % or more). On the other hand, a cleaning composition of thepresent invention comprises hydrotropes in an amount of no more bout 20wt. % (e.g., no more than about 20 wt. %, no more than about 15 wt. %,no more than about 10 wt. %, no more than about 5 wt. %, no more thanabout 1 wt. %). In certain embodiments, the cleaning composition cancomprise hydrotropes in an amount of about 0.01 wt. % to about 20 wt. %(e.g., about 0.02 wt. % to about 18 wt. %, about 0.05 wt. % to about 15wt. %, about 0.1 wt. % to about 10 wt. %, about 1 wt. % to about 5 wt.%), based on the total weight of the cleaning composition.

6) Rheology Modifier

A cleaning composition of the present invention, when in the form of aliquid, can suitably comprise a rheology modifier to provide a matrixthat is “shear-thinning” A shear-thinning fluid, as it is understood bythose skilled in the art, is a fluid the viscosity of which decreases asshear is applied. Thus, at rest, for example, during storage or shippingof a composition, the liquid matrix of the composition preferably has arelatively high viscosity. When shear is applied to the composition,however, such as in the act of pouring or squeezing the composition fromits container, the viscosity of the matrix is lowered to the extent thatdispensing of the fluid product is easily and readily accomplished.

Various materials that are capable of forming shear-thinning fluids whencombined with water or other aqueous liquids are known. One type ofuseful structuring agent for this purpose comprises non-polymeric(except for conventional alkoxylation) crystalline hydroxy-functionalmaterials that can form thread-like structuring systems throughout theliquid matrix when crystallized within the matrix in situ. Suchmaterials include, e.g., crystalline hydroxyl-containing fatty acids,fatty esters, or fatty waxes. Specific examples of crystallinehydroxyl-containing rheology modifiers include castor oil andderivatives. Preferred are hydrogenated castor oil derivatives such ashydrogenated castor oil and hydrogenated castor wax. A number of thesematerials are commercially availalbe, including, for example, THIXCIN®(Elementis Specialties), 1,4-di-O-benzyl-D-Threitol in the R,R, and S, Sforms and any mixtures, optically active or not, and others describedin, for example, U.S. Pat. No. 6,080,708 and International PublicationWO 02/40627.

Suitable polymeric rheology modifiers include those of the polyacrylate,polysaccharide or polysaccharide derivative type. Polysaccharidederivatives typically used as rheology modifiers comprise polymeric gummaterials. Such gums include pectine, alginate, arabinogalactan,carrageenan, gellan gum, xanthan gum and guar gum. Another suitablerheology modifier is a combination of a solvent and a polycarboxylatepolymer. The solvent can be, for example, an alkylene glycol, morepreferably dipropy glycol. For example, the solvent can comprise amixture of dipropyleneglycol and 1,2-propanediol, with a ratio ofdipropyleneglycol to 1,2-propanediol being about 3:1 to about 1:3 (e.g.,about 1:1). The polycarboxylate polymer can be, e.g., a polyacrylate,polymethacrylate, or mixtures thereof. The polyacrylate can be acopolymer of unsaturated mono- or di-carbonic acid and 1-30C alkyl esterof the (meth) acrylic acid, or a polyacrylate of unsaturated mono- ordi-carbonic acid and 1-30C alkyl ester of the (meth) acrylic acid. Someof these polymers are commercially available, for example, under thetradename Carbopol® Aqua 30 (Lubrizol, Wickliffe, Ohio).

The rheology modifiers can be present at a level of about 0.5 wt. % toabout 15 wt. % (e.g., about 1 wt. % to about 12 wt. %, about 2 wt. % toabout 9 wt. %), based on the total weight of the cleaning composition.The polycarboxylate polymer is suitably present at a level of about 0.1wt. % to about 10 wt. % (e.g., about 1 wt. % to about 8 wt. %, about1.5% to about 6 wt. %, about 2 wt. % to about 5 wt. %) in the cleaningcomposition.

6) Solvents or Solvent Systems

A cleaning composition of the invention can be in a liquid form, whereinone or more suitable solvents or solvent systems are included. Suitablesolvents include water, lipophilic fluids, or organic solvents. Examplesof suitable lipophilic fluids include siloxanes, other types ofsilicones, hydrocarbons, glycol ethers, glycerine derivatives such asglycerine ethers, perfluorinated amines, perfluorinated andhydrofluoroether solvents, low-volatility nonfluorinated organicsolvents, diol solvents, other environmentally-friendly solvents andmixtures. Particularly suitable solvents include low molecular weightprimary and secondary alcohols, such as methanol, ethanol, propanil, orisopropanol. Monohydric alcohols, e.g., polyols containing about 2 toabout 6 carbon atoms, and/or about 2 to about 6 hydroxy groups (e.g.,propylene glycol, ethylene glycol, glycerin, and 1,2-propanediol) arealso suitable.

Solvents can be absent, for example, from anhydrous solid embodiments ofthe cleaning compositions of the invention. But in a liquid cleaningcomposition, they are typically present at levels of bout 0.1 wt. % toabout 98 wt. % (e.g., about 1 wt. % to about 90 wt. %, about 10 wt. % toabout 80 wt. %, about 20 wt. % to about 75 wt. %).

7) Organic Sequestering Agent

A cleaning composition of the invention can optionally comprise about0.01 wt. % to about 1.0 wt. % of an organic sequestering agent.Non-limiting example of organic sequestering agent include nitriloaceticacid, EDTA, organic phosphonates, sodium citrate, sodium tartratemonosuccinate, sodium tartrate disuccinate, and mixture thereof.

Certain adjuncts are particularly suitable for laundry/householdcleaning applications as compared to for personal/beauty care cleaningcompositions, while other adjuncts are vise versa. Certain adjuncts arecategorized and described below as particularly suitable for the formeror the latter, but that categorization is not meant to be exclusive inthat adjuncts that are suitable for laundry/household cleaningcompositions can be included in personal/beauty care cleaningcompositions and vise versa as appropriate.

Adjuncts Particularly Suitable for Laundry/Household Applications

1) Bleach System

A bleach system suitable for use herein can contain one or morebleaching agents. Suitable bleaching agents include, e.g., catalyticmetal complexes, activated peroxygen sources, bleach activators, bleachboosters, photobleaches, bleaching enzymes, free radical initiators, andhyohalite bleaches. Suitable activated peroxygen sources include, e.g.,preformed peracids, a hydrogen peroxide source with a bleach activator,or a mixture thereof. Suitable preformed peracids include, e.g.,percarboxylic acids and salts, percarbonic acids and salts, perimidicacids and salts, peroxymonosulfuric acids and salts, and mixturesthereof. Suitable sources of hydrogen peroxide include, e.g., perboratecompounds, percarbonate compounds, perphosphate compounds and mixtures.Suitable types and levels of activated peroxygen sources are describedin, e.g., U.S. Pat. Nos. 5,576,282, 6,306,812, and 6,326,348.

A household cleaning composition of the invention can optionallycomprise photobleach, which can be, for example, a xanthene dyephotobleach, a photo-initiator, or mixtures thereof. Suitablephotobleaches can also catalytic photobleaches and photo-initiators. Incertain embodiments, catalytic photobleaches are selected from the groupconsisting of water soluble phthalocyanines of the formula:

wherein: PC is the phthalocyanine ring system; Me is Zn; Fe(II); Ca; Mg;Na; K; Al—Z₁; Si(IV); P(V); Ti(IV); Ge(IV); Cr(VI); Ga(III); Zr(IV);In(III); Sn(IV) or Hf(VI); Z₁ is a halide; sulfate; nitrate;carboxylate; alkanolate; or hydroxyl ion; q is 0; 1 or 2; r is 1 to 4;Q1 is a sulfur or carboxyl group; or a radical of the formula:—SO₂X₂—R₁—X₃ ⁺; —O—R₁—X₃ ⁺; or —(CH₂), —Y₁ ⁺; in which R₁ is a branchedor unbranched C₁-C₈ alkylene; or 1,3- or 1,4-phenylene; X₂ is —NH—; or—N—C₁-C₅ alkyl; X₃ ⁺ is a group of the formula:

or, in the case where R₁═C₁-C₅ alkylene, also a group of the formula:

Y₁ ⁺ is a group of the formula:

wherein t is 0 or 1; R₂ and R₃ independently of one another are C₁-C₆alkyl; R₄ is C₁-C₅ alkyl; C₅-C₇ cycloalkyl or NR₇R₈; R₅ and R₆independently of one another are C₁-C₅ alkyl; R₇ and R₈ independently ofone another are hydrogen or C₁-C₅ alkyl; R₉ and R₁₀ independently of oneanother are unsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substituted byhydroxyl, cyano, carboxyl, carb-C₁-C₆ alkoxy, C₁-C₆ alkoxy, phenyl,naphthyl or pyridyl; u is from 1 to 6; A₁ is a unit which completes anaromatic 5- to 7-membered nitrogen heterocycle, which may whereappropriate also contain one or two further nitrogen atoms as ringmembers, and B₁ is a unit which completes a saturated 5- to 7-memberednitrogen heterocycle, which may where appropriate also contain 1 to 2nitrogen, oxygen and/or sulfur atoms as ring members; Q₂ is hydroxyl;C₁-C₂₂ alkyl; branched C₃-C₂₂ alkyl; C₂-C₂₂ alkenyl; branched C₃-C₂₂alkenyl and mixtures thereof; C₁-C₂₂ alkoxy; a sulfo or carboxylradical; a radical of the formula:

a branched alkoxy radical of the formula:

an alkylethyleneoxy unit of the formula: -(T₁)d-(CH₂)_(b) (OCH₂CH₂)e-B₃;or an ester of the formula: COOR₁₈, wherein B₂ is hydrogen; hydroxyl;C₁-C₃₀ alkyl; C₁-C₃₀ alkoxy; —CO₂H; —CH₂COOH; —SO₃-M₁OSO₃-M₁; —PO₃ ²⁻M₁;—OPO₃ ²⁻M₁; and mixtures thereof; B₃ is hydrogen; hydroxyl; —COOH;—SO₃-M₁; —OSO₃-M₁ or C₁-C₆ alkoxy; M₁ is a water-soluble cation; T₁ is—O—; or —NH—; X₁ and X₄ independently of one another are —O—; —NH— or—N—C₁-C₅alkyl; R₁₁ and R₁₂ independently of one another are hydrogen; asulfo group and salts thereof; a carboxyl group and salts thereof or ahydroxyl group; at least one of the radicals R₁₁ and R₁₂ being a sulfoor carboxyl group or salts thereof, Y₂ is —O—; —S—; —NH— or—N—C₁-C₅alkyl; R₁₃ and R₁₄ independently of one another are hydrogen;C₁-C₆ alkyl; hydroxy-C₁-C₆ alkyl; cyano-C₁-C₆ alkyl; sulfo-C₁-C₆ alkyl;carboxy or halogen-C₁-C₆ alkyl; unsubstituted phenyl or phenylsubstituted by halogen, C₁-C₄ alkyl or C₁-C₄ alkoxy; sulfo or carboxylor R₁₃ and R₁₄ together with the nitrogen atom to which they are bondedform a saturated 5- or 6-membered heterocyclic ring which mayadditionally also contain a nitrogen or oxygen atom as a ring member;R₁₅ and R₁₆ independently of one another are C₁-C₆ alkyl or aryl-C₁-C₆alkyl radicals; R₁₇ is hydrogen; an unsubstituted C₁-C₆ alkyl or C₁-C₆alkyl substituted by halogen, hydroxyl, cyano, phenyl, carboxyl,carb-C₁-C₆ alkoxy or C₁-C₆ alkoxy; R₁₈ is C₁-C₂₂ alkyl; branched C₃-C₂₂alkyl; C₁-C₂₂ alkenyl or branched C₃-C₂₂ alkenyl; C₃-C₂₂ glycol; C₁-C₂₂alkoxy; branched C₃-C₂₂ alkoxy; and mixtures thereof; M is hydrogen; oran alkali metal ion or ammonium ion, Z₂ ⁻ is a chlorine; bromine;alkylsulfate or arylsulfate ion; a is 0 or 1; b is from 0 to 6; c isfrom 0 to 100; d is 0; or 1; e is from 0 to 22; v is an integer from 2to 12; w is 0 or 1; and A is an organic or inorganic anion, and s isequal to r in cases of monovalent anions A⁻ and less than or equal to rin cases of polyvalent anions, it being necessary for A_(s) ⁻ tocompensate the positive charge; where, when r is not equal to 1, theradicals Q₁ can be identical or different, and where the phthalocyaninering system may also comprise further solubilising groups.

Other suitable catalytic photobleaches include xanthene dyes, sulfonatedzinc phthalocyanine, sulfonated aluminium phthalocyanine, Eosin Y,Phoxine B, Rose Bengal, C. I. Food Red 14, and mixtures. In someembodiment, a photobleach can be a mixture of sulfonated zincphthalocyanine and sulfonated aluminium phthalocyanine, wherein theweight ratio of sulfonated zinc phthalocyanine to sulfonated aluminiumphthalocyanine is greater than 1, greater than 1 but less than about100, or from 1 to about 4.

Suitable photo-initiators include, e.g., aromatic 1,4-quinones such asanthraquinones and naphthaquinones; alpha amino ketones, particularlythose containing benzoyl moieties; alphahydroxy ketones, particularlyalpha-hydroxy acetophenones; phosphorus-containing photoinitiators,including monoacyl, bisacyl and trisacyl phosphine oxide and sulphides;dialkoxy acetophenones; alpha-haloacetophenones; trisacyl phosphineoxides; benzoin and benzoin based photoinitiators; and mixtures thereof.Photo-initiators can, e.g., be 2-ethyl anthraquinone; Vitamin K3;2-sulphate-anthraquinone; 2-methyl 1-[4-phenyl]-2-morpholinopropan-1-one(Irgacure® 907); (2-benzyl-2-dimethylamino-1-(4-morpholinophenyl)-butan-1-one (Irgacure® 369);(1-[4-(2-hydroxyethoxy)-phenyl]-2 hydroxy-2-methyl-1-propan-1-one)(Irgacure® 2959); 1-hydroxy-cyclohexyl-phenyl-ketone (Irgacure® 184)(Ciba); oligo[2-hydroxy 2-methyl-1-[4(1-methyl)-phenyl]propanone(Esacure® KIP 150) (Lamberti);2-4-6-(trimethyl-benzoyl)diphenyl-phosphine oxide,bis(2,4,6-trimethylbenzoyl)-phenyl-phosphine oxide (Irgacure® 819);(2,4,6 trimethyl benzoyl) phenyl phosphinic acid ethyl ester (Lucirin®TPO-L(BASF)); and mixtures thereof.

A number of photobleaches are commercially available, including thosedescribed above, from, e.g., Aldrich; Frontier Scientific; Ciba; BASF;Lamberti S.p.A; Dayglo Color Corporation; Organic Dyestuffs Corp.

2) Pearlescent Agents

Pealescent agents are optional but commonly included ingredients of anumber of household cleaners, especially, e.g., in hard surfacecleaners. They are typically crystalline or glassy solids, transparentor translucent compounds capable of reflecting and/or refracting lightto produce a perlescent effects. For example, they are crystallineparticles insoluble in the composition in which they are incorporated.Preferably the pearlescent agents have the shape of thin plates orspheres (which are generally spherical). As commonly practiced in theart, particle sizes are measured across the largest diameter of spheres.Plate-like particles are defined as those wherein the two dimensions ofthe particle (length and width) are at least 5 times the third dimension(depth or thickness). Other crystal shapes like cubes or needlestypically do not display pearlescent effect and thus are not used asperlescent agents.

Suitable pearlescent agents have D0.99 (sometimes referred to as D99)volume particle size of less than 50 μm. Preferably the pearlescentagents have D0.99 of less than 40 μm, e.g., less than 30 μm. Morepreferably the particles have volume particle size of greater than 1 μm.The D0.99 is a measure of particle size relating to particle sizedistribution and meaning in this instance that 99% of the particles havevolume particle size of less than 50 μm. Volume particle size andparticle size distribution can be measured using conventional methodsand equipment, such as, e.g., a Hydro 2000G (Malvern Instruments). Thechoice of a particle size needs to balance the ease of distribution vs.the efficacy of the pearlescent agent because the smaller the particles,the easier they are suspended, but the lower the efficacy.

Liquid compositions containing less water and more organic solvents willtypically have a refractive index that is higher in comparison to themore aqueous compositions. In these compositions, pearlescent agentswith high refractive index are preferably included because otherwise thepearlescent agents do not impart sufficient visual perlescence even whenintroduced at high levels (e.g., more than about 3 wt. %). In liquidcompositions containing less water and more organic solvents, theperlescent agent is preferably one having a refractive index of morethan 1.41 (e.g., more than 1.8, more than 2.0. In some embodiments, thedifference in refractive index between the pearlescent agent and thecleaning composition or medium, to which pearlescent agent is added, isat least 0.02, or at least 0.2, or at least 0.6.

A liquid cleaning composition may comprise about 0.01 wt. % or more(e.g., about 0.02 wt. % or more, about 0.05 wt. % or more, about 0.1 wt.% or more, about 0.5 wt. % or more, about 1.0 wt. % or more, about 1.5wt. % or more) of one or more pearlescent agents. Typically, however,the liquid composition comprises no more than about 2 wt. % (e.g., nomore than about 1.5 wt. %, no more than about 1.0 wt. %, no more thanabout 0.5 wt. %) of one or more pearlescent agents. For example, aliquid cleaning composition herein comprises about 0.01 wt. % to about2.0 wt. % (e.g., about 0.1 wt. % to about 1.5 wt. %) of pearlescentagents.

Suitable pearlescent agents may be organic or inorganic. Organicpearlescent agents include, e.g., monoester and/or diester of alkyleneglycols, propylene glycol, diethylene glycol, dipropylene glycol,methylene glycol or tetraethylene glycol with fatty acids containing 6to 22, preferably about 12 to about 18 carbon atoms, e.g., caproic acid,caprylic acid, 2-ethyhexanoic acid, capric acid, lauric acid,isotridecanoic acid, myristic acid, palmitic acid, palmitoleic acid,stearic acid, isostearic acid, oleic acid, elaidic acid, petroselicacid, linoleic acid, linolenic acid, arachic acid, gadoleic acid,behenic acid, erucic acid, and mixtures.

Inorganic pearlescent agents include mica, metal oxide coated mica,silica coated mica, bismuth oxychloride coated mica, bismuthoxychloride, myristyl myristate, glass, metal oxide coated glass,guanine, glitter, and mixtures thereof.

Organic pearlescent agent such as ethylene glycol mono stearate andethylene glycol distearate provide pearlescence, but typically only whenthe composition is in motion. Hence only when the composition is pouredwill the composition exhibit pearlescence. Inorganic pearlescentmaterials are preferred as the provide both dynamic and staticpearlescence. By dynamic pearlescence it is meant that the compositionexhibits a pearlescent effect when the composition is in motion. Bystatic pearlescence it is meant that the composition exhibitspearlescence when the composition is static.

Inorganic pearlescent agents are available as a powder, or as a slurryof the powder in an appropriate suspending agent. Suitable suspendingagents include ethylhexyl hydroxy-stearate, hydrogenated castor oil. Thepowder or slurry of the powder can be added to the composition withoutthe need for any additional process steps.

Optionally, co-crystallizing agents can be used to enhance thecrystallization of the organic pearlescent agents. Suitableco-crystallizing agents include but are not limited to fatty acidsand/or fatty alcohols having a linear or branched, optionally hydroxylsubstituted, alkyl group containing from about 12 to about 22,preferably from about 16 to about 22, and more preferably from about 18to 20 carbon atoms, such as palmitic acid, linoleic acid, stearic acid,oleic acid, ricinoleic acid, behenyl acid, cetearyl alcohol,hydroxystearyl alcohol, behenyl alcohol, linolyl alcohol, linolenylalcohol, and mixtures thereof.

3) Perfumes/Fragrances

The term “perfume” as used herein encompasses individual perfumeingredients as well as perfume accords. The perfume ingredients areoften premixed to form a perfume accord prior to adding to a cleaningcomposition. As used herein, the term “perfume” can also include perfumemicroencapsulates. Perfume microcapsules comprise perfume raw materialsencapsulated within a capsule made with materials selected from urea andformaldehyde; melamine and formaldehyde; phenol and formaldehyde;gelatine; polyurethane; polyamides; cellulose ethers; cellulose esters;polymethacrylate; and mixtures thereof. Encapsulation techniques areknown and described in, for example, “Microencapsulation”: methods andindustrial applications, Benita & Simon, eds. (Marcel Dekker, Inc.,1996).

The perfume ingredients that can be included in a cleaning compositioncan include various natural and synthetic chemicals. Exemplary perfumeingredients include aldehydes, ketones, esters, natural extracts,natural essences and the like.

Industrial cleaning compositions often do not comprise perfumeingredients. However, perfume ingredients are commonly found inhousehold and personal care cleaning compositions. When present, thelevel of perfume or perfume accord is, e.g., present in an amount ofabout 0.0001 wt. % or more (e.g., about 0.01 wt. % or more, about 0.1wt. % or more, about 0.5 wt. % or more, about 2 wt. % or more), based onthe total weight of the cleaning composition. For example, the level ofperfume or perfume accord can be present in an amount of about 0.0001wt. % to about 10 wt. % (e.g., about 0.01 wt. % to about 5 wt. %, about0.1 wt. % to about 2 wt. %, preferably about 0.02 wt. % to about 0.8 wt.%, or about 0.003 wt. % to about 0.6 wt. %), by weight of the detergentcomposition. The level of perfume ingredients in a perfume accord, ifone exists, is typically from about 0.0001 wt. % to about 99 wt. % byweight of the perfume accord. Exemplary perfume ingredients and perfumeaccords are disclosed in, for example, U.S. Pat. Nos. 5,445,747,5,500,138, 5,531,910, 6,491,840, and 6,903,061.

4) Dyes, Colorants, and Preservatives

The cleaning compositions herein can optionally contain dyes, colorants,and/or preservatives, or contain one or more, or none of thesecomponents. The dyes, colorants and/or preservatives can be naturallyoccurring or slightly processed from natural materials, or they can besynthetic. For example, natural-occurring preservatives include benzylalcohol, potassium sorbate and bisabalol, sodium benzoate, and2-phenoxyethanol. Synthetic preservatives can be selected from, forexample, mildewstate or bacteriostate, methyl, ethyl, and propylparabens, bisguarnidine components (e.g., Dantagard™ and/or Glydant™(Lonza Group)). Midewstate or bacteriostate compounds include, withoutlimitation, KATHON® GC, a 5-chloro-3-methyl-4-isothiazolin-3-one,KATHON® ICP, a 2-methyl-4-isothiazolin-4-one, and a blend thereof, andKATHON® 886, a 5-chloro-2-methyl-4-isothazolin-3-one (Dow Chemicals);BRONOPOL, a 2-bromo-2-nitropropane 1, 3 diol (Boots, Co. Ltd.);DOWICIDE™ A, a 1,2-benzoisothiazolin-3-one (Dow Chemicals); and IRGASAN®DP 200, a 2,4,4′-trichloro-2-hydroxydiphenylether (Ciba-Geigy, AG).

Dyes and colorants include synthetic dyes such as Liquitint® Yellow orBlue or natural plant yes or pigments, such as natural yellow, orange,red, and/or brown pigment, such as carotenoids, including, for example,beta-carotene and lycopene. The composition can additionally containfluorescent whitening agents or bluing agents. Certain dyes can be lightsensitive, including for example Acid Blue 145 (Crompton), Hidacid® blue(Hilton, Davis, Knowles & Triconh); Pigment Green No. 7, FD&C Green No.7, Acid Blue 1, Acid Blue 80, Acid Violet 48, and Acid Yellow 17 (SandozCorp.); D&C Yellow No. 10 (Warner Jenkinson).

If present, dyes or colorants are, e.g., present in an amount of about0.001 wt. % or more (e.g., about 0.002 wt. % or more, 0.01 wt. % ormore, 0.05 wt. % or more, 0.1 wt. % or more; 0.5 wt. % or more).Usually, dyes and colorants are present, if at all, in an amount of nomore than about 1 wt. % (e.g., no more than about 0.8 wt. %, no morethan about 0.5 wt. %, no more than about 0.2 wt. %, no more than about0.1 wt. %, no more than about 0.01 wt. %). For example, dyes andcolorants can be present in a cleaning composition herein in an amountof about 0.001 wt. % to about 1 wt. % (e.g., about 0.01 wt. % to about0.4 wt. %).

5) Fabric Care Benefit Agents

A household cleaning composition can be a laundry detergent, wherein apreferred optional ingredient can be a fabric care benefit agent. Asused herein, “fabric care benefit agent” refers to any material that canprovide fabric care benefits such as fabric softening, color protection,pill/fuzz reduction, anti-abrasion, anti-wrinkle, and the like togarments and fabrics, particularly on cotton and cotton-rich garmentsand fabrics, when an adequate amount of the material is present on thegarment/fabric. Non-limiting examples of fabric care benefit agentsinclude cationic surfactants, silicones, poly olefin waxes, latexes,oily sugar derivatives, cationic polysaccharides, polyurethanes andmixtures. Suitable silicones include, e.g., silicone fluids such aspoly(di)alkyl siloxanes, especially polydimethyl siloxanes and cyclicsilicones.

Polydimethyl siloxane derivatives include, e.g., organofunctionalsilicones. One embodiment of functional silicone are the ABn typesilicones, as described in U.S. Pat. Nos. 6,903,061, 6,833,344, andInternational Publication WO02/018528. A number of silicones arecommercially available, including, e.g., Waro™ and Silsoft™ 843 (GESilicones). Functionalized silicones or copolymers with one or moredifferent types of functional groups such as amino, alkoxy, alkyl,phenyl, polyether, acrylate, silicon hydride, mercaptoproyl, carboxylicacid, quaternized nitrogen are also suitable as fabric care benefitagents. A number of these are commercially available including, e.g.,SM2125, Silwet 7622 (GE Silicones), DC8822, PP-5495, DC-5562 (DowChemicals), KF-888, KF-889 (Shin Etsu Silicones); Ultrasil® SW-12,Ultrasil® DW-18, Ultrasil® DW-AV, Ultrasil® Q-Plus, Ultrasil® Ca-I,Ultrasil® CA-2, Ultrasil® SA-I, Ultrasil® PE-100 (Noveon Inc.), Pecosil®CA-20, Pecosil® SM-40, Pecosil® PAN-150 (Phoenix Chemical). Oily sugarderivatives suitable as fabric care benefit agents were described inInternational Publication WO 98/16538. Olean® is a commercial brand forcertain oily sugar derivatives marketed by The Procter and Gamble Co.

Many dispersible polyolefins can be used to provide fabric carebenefits. The polyolefins can be in the form of waxes, emulsions,dispersions, or suspensions. Preferably, the polyolefin is apolyethylene, polypropylene, or a mixture. The polyolefin can bepartially modified to contain various functional groups, such ascarboxyl, alkylamide, sulfonic acid or amide groups. For example, thepolyolefin is partially carboxyl modified or oxidized.

Polymer latex can also be used to provide fabric care benefits in awater based cleaning composition. Non-limiting examples of polymerlatexes include those described in, e.g., International Publication WO02/018451. Additional non-limiting examples include the monomers used inproducing polymer latexes, such as 100% or pure butylacrylate,butylacrylate and butadiene mixtures with at least 20 wt. % ofbutylacrylate, butylacrylate and less than 20 wt. % of other monomersexcluding butadiene, alkylacrylate with an alkyl carbon chain at orgreater than C₆, alkylacrylate with an alkyl carbon chain at or greaterthan C₆ and less than 50 wt. % of other monomers, or a third monomeradded into monomer systems above.

Cationic surfactants are also useful in this invention. Examples ofcationic surfactants have been described in, e.g., U.S. PatentPublication US2005/0164905.

Fatty acids can also be used as fabric care benefit agents. Whendeposited on fabrics, fatty acids or soaps thereof, provide fabric carebenefits (e.g., softness, shape retention) to laundry fabrics. Usefulfatty acids (or soaps, such as alkali metal soaps) are the higher fattyacids containing from about 8 to about 24 carbon atoms, more preferablyfrom about 12 to about 18 carbon atoms. Soaps can be made by directsaponification of fats and oils or by the neutralization of free fattyacids. Particularly useful are the sodium and potassium salts of themixtures of fatty acids derived from coconut oil and tallow. Fatty acidscan be from natural or synthetic origin, both saturated and unsaturatedwith linear or branched chains.

Color care agents are another type of fabric care benefit agent that canbe suitably included in a cleaning composition. Examples include metallocatalysts for color maintenance, such as those described inInternational Publication WO 98/39403.

Fabric care benefit agents, when present in a household cleaningcomposition such as a laundry detergent composition, can suitably bepresent at a level of up to about 30 wt. % (e.g., up to about 20 wt. %,up to about 15 wt. %, up to about 10 wt. %, up to about 5 wt. %, up toabout 2 wt. %), based on the total weight of the cleaning composition.For example, a cleaning composition of the invention comprises about 1wt. % to about 20 wt. % (e.g., about 2 wt. % to about 15 wt. %, about 5wt. % to about 10 wt. %) of one or more fabric care benefit agents.

6) Deposition Aid

As used herein, “deposition aid” refers to any cationic polymer orcombination of cationic polymers that significantly enhance thedeposition of the fabric care benefit agent onto the fabric duringlaundering. An effective deposition aid typically has a strong bindingcapability with the water insoluble fabric care benefit agents viaphysical forces such as van der Waals forces or non-covalent chemicalbonds such as hydrogen bonding and/or ionic bonding.

An exemplary deposition aid is a cationic or amphoteric polymer.Amphoteric polymers have a net cationic charge. The cationic chargedensity of the polymer can range from about 0.05 milliequivalents/g toabout 6 milliequivalents/g. The charge density is calculated by dividingthe number of net charge per repeating unit by the molecular weight ofthe repeating unit. Nonlimiting examples of deposition aids includecationic polysaccharides, chitosan and its derivatives, and cationicsynthetic polymers. Specific deposition aids include, e.g., cationichydroxy ethyl cellulose, cationic starch, cationic guar derivatives, andmixtures. Certain deposition aids are commercially available, including,e.g., the JR 30M, JR 400, JR 125, LR 400 and LK 400 polymers (AmercholCorp.), Celquat® H200, Celquat® L-200, and the Cato® starch (NationalStarch and Chemical Co.), and Jaguar Cl 3 and Jaguar Excel (Rhodia,Inc.).

7) Fabric Substantive and Hueing Dye

Dyes can be included in a cleaning composition of the invention, e.g., alaundry detergent. Conventionally, dyes include certain types of acid,basic, reactive, disperse, direct, vat, sulphur or solvent dyes. Forinclusion in cleaning compositions, direct dyes, acid dyes, and reactivedyes are preferred. Direct dyes are water-soluble dyes taken up directlyby fibers from an aqueous solution containing an electrolyte, presumablydue to selective adsorption. In the Color Index system, direct dyerefers to various planar, highly conjugated molecular structures thatcontain one or more anionic sulfonate group. Acid dyes are water solubleanionic dyes that are applied from an acidic solution. Reactive dyes arethose containing reactive groups capable of forming covalent linkageswith certain portions of the molecules of natural or synthetic fibers.Suitable fabric substantive dyes that can be included in a cleaningcomposition include, e.g., an azo compound, stilbenes, oxazines andphthalocyanines.

Hueing dyes are another type of dyes that may be present in a householdcleaning composition of the invention. Such dyes have been found toexhibit good tinting efficiency during a laundry wash cycle withoutexhibiting excessive undesirable build up during laundering. Typically,a hueing dye is included in the laundry detergent composition in anamount sufficient to provide a tinting effect to fabric washed in asolution containing the detergent. In one embodiment, the detergentcomposition comprises, e.g., about 0.0001 wt. % to about 0.05 wt. %(e.g., about 0.001 wt. % to about 0.01 wt. %) of a hueing dye.

8) Dye Transfer Inhibitors

A household cleaning composition of the invention, e.g., a laundrydetergent composition, can comprise one or more compounds for inhibitingdye transfer from one fabric to another of solubilized and suspendeddyes encountered during fabric laundering operations involving coloredfabrics. Exemplary dye transfer inhibitors include polymedc dye transferinhibiting agents, which are capable of complexing or absorbing thefugitive dyes washed out of dyed fabrics before the dyes have anopportunity to become attached to other articles in the wash. Polymedcdye transfer agents are described in, e.g., International Publication WO98/39403. Modified polyethyleneimine polymers, such as those describedin International Publication WO 00/05334, which are water-soluble ordispersible, modified polyamines can also be used. Other exemplary dyetransfer inhibiting agents include, e.g., polyvinylpyrridine N-oxide(PVNO), polyvinyl pyrrolidone (PVP), polyvinyl imidazole,N-vinyl-pyrrolidone and N-vinylimidazole copolymers (PVPVI), copolymersthereof, and mixtures. The amount of dye transfer inhibiting agents inthe cleaning composition can be, e.g., about 0.01 wt. % to about 10 wt.% (e.g., about 0.02 wt. % to about 5 wt. %, about 0.03 wt. % to about 2wt. %).

9) Optional Ingredients

Unless specified herein below, an “effective amount” of a particularadjunct or ingredient is preferably present in an amount of about 0.01wt. % or more (e.g., about 0.1 wt. % or more, about 0.5 wt. % or more,about 1.0 wt. % or more, about 2.0 wt. % or more), based on the totalweight of the detergent composition. Optional adjuncts are usuallypresented in an amount of no more than about 20 wt. % (e.g., no morethan about 15 wt. %, no more than about 10 wt. %, no more than about 5wt. %, or no more than about 1 wt. %).

Examples of other suitable cleaning adjuncts, one or more of which maybe included in a cleaning composition, include, e.g., effervescentsystems comprising hydrogen peroxide and catalase; optical brightenersor fluorescers; soil release polymers; dispersants; suds suppressors;photoactivators; hydrolysable surfactants; preservatives; anti-oxidants;anti-shrinkage agents; gelling agents (e.g., amidoamines, amidoamineoxides, gellan gums); anti-wrinkle agents; germicides; fungicides; colorspeckles; antideposition agents such as celluose derivatives, coloredbeads, spheres or extrudates; sunscreens; fluorinated compounds; clays;luminescent agents or chemiluminescent agents; anti-corrosion and/orappliance protectant agents; alkalinity sources or other pH adjustingagents; solubilizing agents; processing aids; pigments; free radicalscavengers, and mixtures. Suitable materials and effective amounts aredescribed in, e.g., U.S. Pat. Nos. 5,705,464, 5,710,115, 5,698,504,5,695,679, 5,686,014 and 5,646,101. Mixtures of the above components canbe made in any proportion.

10) Encapsulated Composition

A cleaning composition, such as a household cleaning compositionincluding a laundry detergent, a dishwashing liquid, or a surfacecleaning composition, of the present invention can optionally beencapsulated within a water soluble film. The water-soluble film can bemade from polyvinyl alcohol or other suitable variations, carboxy methylcellulose, cellulose derivatives, starch, modified starch, sugars, PEG,waxes, or combinations thereof.

In certain embodiment the water-soluble film may comprise other adjunctssuch as copolymer of vinyl alcohol and a carboxylic acid, the advantagesof which have been desbribed in, for example, U.S. Pat. No. 7,022,656.An exemplary benefit of such encapsulation practice is the improvementof the shelf-life of the pouched composition. Another exemplaryadvantage is that this practice provides improved cold water (e.g., lessthan 10° C.) solubility to the cleaning composition. The level of theco-polymer in the film material is at least about 60 wt. % (e.g., about65 wt. %, about 70 wt. %, about 80 wt. %) by weight. The polymer canhave any average molecular weight, preferably about 1,000 daltons to1,000,000 daltons (e.g., about 10,000 daltons to about 300,000 daltons,about 15,000 daltons to 200,000 daltons, about 20,000 daltons to 150,000daltons). In certain embodiments, the copolymer present in the film isabout 60% to about 98% hydrolysed (e.g., about 80% to 95% hydrolysed),to improve the dissolution of the material. In certain embodiments, thecopolymer comprises about 0.1 mol % to about 30 mol % (e.g., about 1 mol% to about 6 mol %) of carboxylic acid. In certain embodiments, thewater-soluble film comprises additional co-monomers, including, forexample, sulfonates and ethoxylates such as2-acrylamido-2-methyl-1-propane sulphonic acid. In further embodiments,the film can also comprise other ingredients, including, for example,plasticizers, for example, glycerol, ethylene glycol, diethyleneglycol,propane diol, 2-methyl-1,3-propane diol, sorbitol, and mixtures thereof,additional water, disintegrating aids, fillers, anti-foaming agents,emulsifying/dispersing agents, and/or antiblocking agents. It may beuseful that the pouch or water-soluble film itself comprises a detergentadditive to be delivered to the wash water, for example organicpolymeric soil release agents, dispersants, dye transfer inhibitors.Optionally the surface of the film of the pouch may be dusted with finepowder to reduce the coefficient of friction. Sodium aluminosilicate,silica, talc and amylose are examples of suitable fine powders. Certainwater-soluble films are commercially available, for example, thosemarketed under the tradename M8630™ (Mono-Sol).

Adjuncts Particularly Suitable for Personal Care Applications

1) Hair Conditioning Agents

Cleaning compositions of the invention can comprise, in some embodimentssuch as, for example, in personal or beauty care applications, certainknown conditioning agents. An exemplary conditioning agent especiallysuitable for personal care compositions such as shampoos, is a siliconeor a silicone-containing material. Such materials can be selected from,e.g., non-volatile silicones, siloxane gums and resins, aminofunctionalsilicones, quaternary silicones, and mixtures thereof with each otherand with volatile silicones. Examples of these silicone polymers havebeen disclosed, for example, in U.S. Pat. No. 6,316,541.

Silicone oils are flowable silicone materials having a viscosity asmeasured at 25° C. of less than about 50,000 centistokes (e.g., lessthan aobut 30,000 centistokes). For example, silicone oils typicallyhave a viscosity of about 5 centistokes to about 50,000 centistokes(e.g., about 10 centistokes to about 30,000 centistokes). Suitablesilicone oils include polyalkyl siloxanes, polyaryl siloxanes,polyalkylaryl siloxanes, polyether siloxane copolymers, and mixtures.Other insoluble, non-volatile silicone fluids having hair conditioningproperties can also be used. Methods of making microemulsions ofsilicone particles are described in the art, including, e.g., thetecnique described in U.S. Pat. No. 6,316,541. The silicone may, e.g.,be a liquid at ambient temperatures, so as to be of a suitable viscosityto enable the material itself to be readily emulsified to the requiredparticle size of about 0.15 microns or less.

The amount of silicone incorporated into a cleaning composition of theinvention may depend on the type of composition and the particularsilicone materials used. A preferred amount is about 0.01 wt. % to about10 wt. %, although these limits are not absolute. The lower limit isdetermined by the minimum level to achieve acceptable conditioning for atarget consumer group and the upper limit by the maximum level to avoidmaking the hair and/or skin unacceptably greasy. The activity of themicroemulsion can be adjusted accordingly to achieve the desired amountof silicone or a lower level of the preformed microemulsion can beadded.

The microemulsion of silicone oil may be further stabilized by sodiumlauryl sulfate or sodium lauryl ether sulfate with 1-10 moles ofethoxylation. Additional emulsifier, preferably chosen from anionic,cationic, nonionic, amphoteric and zwitterionic surfactants, andmixtures thereof may be present. The amount of emulsifier will typicallybe in the ratio of about 1:1 to about 1:7 parts by weight of thesilicone, although larger amounts of emulsifier can be used, e.g., inabout 5:1 parts by weight of the silicone or more. Use of theseemulsifiers may be necessary to maintain clarity of the microemulsion ifthe microemulsion is diluted prior to addition to the personal carecleaning composition. The same detersive surfactants in the cleaningcomposition can also serve as the emulsifier in the preformedmicroemulsion.

The silicone microemulsion may be further stabilized using an emulsionpolymerization process. A suitable emulsion polymerization process hasbeen described by, for example, U.S. Pat. No. 6,316,541. A typicalemulsifier is TEA dodecyl benzene sulfonate which is formed in theprocess when triethanolamine (TEA) is used to neutralize the dodecylbenzene sulfonic acid used as the emulsion polymerization catalyst. Ithas been found that selection of the anionic counterion, typically anamine, and/or selection of the alkyl or alkenyl group in the sulfonicacid catalyst can further improve the stability of the microemulsion inthe shampoo composition. Examples of preferred amines include, withoutlimitation, triisopropanol amine, diisopropanol amine, and aminomethylpropanol.

2) Pearlescent Agents

Pearlescent agents, such as those described herein above can be suitablyincluded in a personal care cleaning composition such as a shampoo. Theyare defined, for the purpose of the present disclosure, as materialswhich impart to a composition the appearance of mother of pearl.Pearlescence is produced by specular reflection of light. Lightreflected from pearl platelets or spheres as they lie essentiallyparallel to each other at different levels in the composition creates asense of depth and luster. Some light is reflected off the pearlescentagent, and the remainder will pass through the agent, which may passdirectly through or be refracted. Reflected, refracted light produces adifferent colour, brightness and luster.

3) Cationic Cellulose or Guar Polymer

Cleaning compositions of the present invention can further contain acationic polymer to aid the deposition of the silicone oil component andenhance conditioning performance. Non limiting examples of such polymersare described in the CTFA Cosmetic Ingredient Dictionary, 3rd ed,Estrin, Crosley, & Haynes eds., (The Cosmetic, Toiletry, and FragranceAssociation, Inc., Washington, D. C. (1982)). Suitable cataionicpolymers include polysaccharide polymers, such as cationic cellulosederivatives, for example, salts of hydroxyethyl cellulose reacted withtrimethyl ammonium substituted epoxide, referred to in the industry(CTFA) as Polyquaternium 10, as well as Polymer LR, JR, JP and KG seriespolymers (Amerchol Corp.). Other suitable cationic cellulose polymersincludes the polymeric quaternary ammonium salts of hydroxyethylcellulose reacted with lauryl dimethyl ammonium-substituted epoxidereferred to in the industry (CTFA) as Polyquaternium 24, available underthe tradename Polymer LM-200 (Amerchol Corp). Suitable cationic guarpolymers include cationic guar gum derivatives, such as guarhydroxypropyltrimonium chloride, and those described in, for example,U.S. Pat. No. 5,756,720. Certain of these polymers are commercialyavailable, including, for example, Jaguar® Excel (Rhodia Corp.).

When used, the cationic polymers herein are either soluble in thecleaning composition or are soluble in a complex coacervate phase in thecleaning composition formed by the cationic polymer and the anionic,amphoteric and/or zwitterionic detersive surfactant component describedhereinbefore. Complex coacervates of the cationic polymer can also beformed with other charged materials in the composition.

Concentrations of the cationic polymer in the composition can range fromabout 0.01 wt. % to about 3 wt. % (e.g., about 0.05 wt. % to about 2 wt.%, about 0.1 wt. % to about 1 wt. %). Suitable cationic polymers havecationic charge densities of at least about 0.4 meq/gm (e.g., at leastabout 0.6 meq/gm). Suitable cationic polymers have cationic chargedensities of no more than about 5 meq/gm, at the pH of intended use ofthe cleaning composition. In an exemplary personal care cleaningcomposition, such as, for example, a shampoo, which generally has a pHrange of about 3 to about 9 (e.g., about 4 to about 8). As used herein,“cationic charge density” of a polymer refers to the ratio of the numberof positive charges on the polymer to the molecular weight of thepolymer. The average molecular weight of suitable cationic guars andcellulose polymers is typically at least about 800,000 daltons. Forexample, suitable cationic polymers, which can be included in a cleaningcomposition of the present invention, is one of sufficiently highcationic charge density to effectively enhance deposition efficiency ofthe solid particle components in the cleaning composition. Cationicpolymers comprising cationic cellulose polymers and cationic guarderivatives with cationic charge densities of at least about 0.5 meq/gmand preferably less than about 7 meq/gm are suitable.

Preferably, the deposition polymers give good clarity and adequateflocculation on dilution with water during use, especially when suitableelectrolytes including, e.g., sodium chloride, sodium benzoate,magnesium chloride, and magnesium sulfate, are added.

4) Perfumes/Fragrances

Just as perfumes or perfume accords are typically included in ahousehold cleaning composition of the invention, perfumes or perfumeaccords as described herein (e.g., supra) are often included in apersonal care cleaning composition, such as a shampoo or a bodywashcomposition. The perfume ingredients, which optionally can be formulatedinto a perfume accord prior to blending or formulating the cleaningcomposition, can be obtained from a wide variety of natural or syntheticsources. They include, without limitation, aldehydes, ketones, esters,and the like. They also include, for example, natural extracts andessences, which can include complex mixtures of ingredients, such asorange oils, lemon oils, rose extracts, lavender, musk, patchouli,balsamic essence, sandalwood oil, pine oil, cedar, and the like. Theamount of perfume to be included in a cleaning composition of theinvention can vary, for example, from about 0.0001 wt. % to about 2 wt.% (e.g., about 0.01 wt. % to about 1.0 wt. %, about 0.1 wt. % to about0.5 wt. %), based on the total weight of the cleaning composition.

5) Sensory Indicators—Silica Particles

Optionally, in a personal care cleaning composition of the invention,various sensory indicators can be included. These agents provide achange in sensory feel after an appropriate usage time, allowing foreasy and precise recognition for the appropriate time of washing. Forexample, these agents are particularly suitable for cleaningcompositions such as hand cleansers. An exemplary type of sensoryindicators are silica particles. The properties of the silica particlemay be adjusted to provide the desired end point in time.

Various silica particles are commercially available, including, forexample, those made and distributed by INEOS Silicas Ltd. Theseparticles have also been described in, for example, U.S. Pat. No.6,165,510, U.S. Patent Publication 2003/0044442.

Silica particles can be present in an amount that can initially be feltby hands when starting washing with the cleaning composition. In oneembodiment, the amount of silica particles is about 0.05 wt. % to about8 wt. %. In some embodiments, suitable silica particles can have aninitial average diameter of about 50 μm to about 600 μm (e.g., about 180to about 420 μm). In some embodiments, silica particles can furthercomprise color or pigment on the surface. In other embodiments, suitablesilica particles diminish in size and cannot be felt by users duringwashing before about 5 min, about 2 min, about 30 sec, about 25 sec,about 20 sec, about 15 sec, about 10 sec, about 5 sec, about 5 to about30 sec, or about 10 to about 30 sec.

Silica particles can also, in addition to providing sensory indications,improve the dispensing of the cleaning composition. For example, byincluding these particles, the cleaning composition, such as a liquidhand cleaner or a shampoo, may achieve a desirable thickness such thatit is easier to be dispensed with a pump.

It is often desirable to regulate the viscosity of a compositioncomprising silica particles, however. Addition of glycerin has beenfound to be an effective approach to achieve this regulation. Glycerinis typically added to a composition comprising silica particles in anamount of at least about 1 wt. % (e.g., about 2 wt. %, about 2.5 wt. %,about 3 wt. %, about 4 wt. %, about 5 wt. %, or about 6 wt. %), based onthe total weight of the cleaning composition. In some embodiments,glycerin is added in an amount of less than about 10 wt. % (e.g., lessthan about 8 wt. %, less than about 6 wt. %, less than about 4 wt. %,less than about 2 wt. %). The addition of glycerin may, in certainembodiments, help prevent clogging of pumps.

6) Suspension Agents-Viscosity Control

Cleaning compositions of the invention can also include a suspendingagent that allows the particulate matters therein, e.g., the silicaparticles, to remain suspended. Suspending agents are materials that arecapable of increasing the ability of the composition to suspendmaterial. Examples of suspending agents include, e.g., syntheticstructuring agents, polymeric gums, polysaccharides, pectin, alginate,arabinogalactan, carrageen, gellan gum, xanthum gum, guar gum, rhamsangum, furcellaran gum, and other natural gum. An exemplary syntheticstructuring agent is a polyacrylate. An exemplary acrylate aqueoussolution used to form a stable suspension of the solid particles ismanufactured by Lubrizol as CARBOPOL™ resins, also known as CARBOMER™,which are hydrophilic high molecular weight, crosslinked acrylic acidpolymers. Other polymers suitable as suspension agents include, e.g.,CARBOPOL™ Aqua 30, CARBOPOL™ 940 and CARBOPOL™ 934.

The suspending agents can be used alone or in combination. The amount ofsuspending agent can be any amount that provides for a desired level ofsuspending ability. In certain embodiment, the suspending agent ispresent in an amount of about 0.01 wt. % to about 15 wt. % (e.g., about0.1 wt. % to about 12 wt. %, about 1 wt. % to about 10 wt. %, about 2 wt% to about 5 wt. %) by weight of the cleaning composition.

7) Other Suitable Adjuncts

A number of other adjuncts can be suitable for inclusion in a personalcare cleaning composition. Those include, for example, thickeners, suchas hydroxyl ethyl cellulose derivatives (e.g., Methocel™ products, DowChemicals; Natrosol® products, Aqualon Ashland; Carbopol™ products,Lubrizol).

Stability enhancers can also be included as suitable adjuncts. They aretypically nonionic surfactants, including those having anhydrophilic-lipophilic balance range of about 9-18. These surfactantscan be straight chained or branched chained, and they typicallycontaining various levels of ethoxylation/propoxylation. The nonionicsurfactants useful in the present invention are preferably formed from afatty alcohol, a fatty acid, or a glyceride with a C₃ to C₂₄ carbonchain, preferably a C₁₂ to C₁₈ carbon chain derivatized to yield aHydrophilic-Lipophilic Balance (HLB) of at least 9. HLB is understood tomean the balance between the size and strength of the hydrophilic groupand the size and strength of the lipophilic group of the surfactant.Suitable adjuncts for personal care cleaning compoisitons can alsoinclude various vitamins, including, for example, vitamin B complex;incuding thiamine, nicotinic acid, biotin, pantothenic acid, choline,riboflavin, vitamin B6, vitamin B12, pyridoxine, inositol, carnitine,vitamins A, C, D, E, K, and their derivatives.

Further suitable adjuncts may include one or more materials such asantimicrobial agents, antifungal agents, antidandruff agents, dyes, foamboosters, pediculocides, pH adjusting agents, preservatives, proteins,skin active agents, sunscreens, UV absorbers, minerals,herbal/fruit/food extracts, sphigolipid derivatives or syntheticderivatives, and clay.

Examples of Preferred Embodiments

Surfactant compositions of the invention can be formulated or usedwithout substantial post-production processing. This is especially thecase if the surfactant composition is applied in industrial settings,for example, in oil industry for oil recovery applications. Becausetypically minimum purity specification is required in such settings, itis potentially possible to use whole-cell broths. Surfactants comprisingmicrobially-produced branched fatty alcohols and derivatives prepared inaccordance with the methods herein are relatively more selective, ascompared with conventional chemical surfactants. As such, they arerequired in small quantities, and effective under a broad range of oiland reservoir conditions. They are also more environmentally friendly inprotection of coastal areas from additional damage inflicted bysynthetic chemicals, because they are readily biodegradable and havelower toxicity than synthetic surfactants. Potentially an about 30% ormore increase in total oil recovery from underground sandstone can beachieved using surfactants comprising microbially produced fattyalcohols and derivatives such as those described herein.

Microbially-produced fatty alcohols, including branched fatty alcoholsand derivatives thereof such as those described herein, are also moreanaerobic, halotolerant and thermo-tolerant as compared to theirpetroleum-derived counterparts, making surfactants comprising thesefatty alcohols particularly useful for in situ enhanced oil recovery.These surfactants are potent reducers of oil viscosity, making it vastlyeasier to pump heavy oils from underground sandstone as well as throughcommercial pipelines for long distances. Microbially-produced fattyalcohols and derivatives and surfactants comprising these materials canalso be used to desludge crude oil storage tanks. The branched fattyalcohols and derivates described herein also have improved lowtemperature properties, and are thus particularly suited for applicationin low temperature environments such as in the deep sea.

Potentially, suitable host cells can be engineered such that the culturebroth not only provide suitable surfactants but also providesbiodegradation of hydrocarbons, resulting in microbial remediation ofhydrocarbon- and crude oil-contaminated soils. Furthermore, the branchedfatty alcohols, derivatives thereof, as well as the surfactantscomprising these materials can be used to manage and emulsifyhydrocarbon-water mixtures. This capacity to effectively emulsifyoil/water mixtures can be utilized in oil spill management.

With more extensive post-production processing, surfactants comprisingthe branched fatty alcohols and derivatives as described herein can beparticularly suitable as food additives or in the health care andcosmetic industries. The branching of these molecules confer addedoxidative stability and significantly decreased volatility and vaporpressure. They are also useful as ingredients in various household andpersonal and/or pet care cleaning compositions, with particularadvantages at lower washing temperatures.

In certain embodiments, the invention features a surfactant compositioncomprising about 0.001 wt. % to about 100 wt. % (e.g., about 0.01 wt. %to about 80 wt. %, about 0.1 wt. % to about 70 wt. %, about 1 wt. % toabout 60 wt. %, about 5 wt. % to about 50 wt. %) of one or moremicrobially produced branched fatty alcohols and/or derivatives thereof.An exemplary surfactant composition of the invention comprises about 0.1wt. % to about 50 wt. % of microbially produced branched fatty alcoholsand/or derivatives thereof. The surfactant composition of the presentinvention can further comprise one or more other co-surfactants, derivedfrom similar origins (e.g., microbially produced) or different origins(e.g., chemically synthesized, derived from petroleum sources).

In another aspect, the invention pertains to a cleaning compositioncomprising one or more surfactants comprising branched fatty alcoholsand derivatives produced in accordance with the methods describedherein. The inventive cleaning composition can be formulated as a solidcleaning composition or as a liquid cleaning composition.

In certain embodiment, the invention provides a cleaning compositioncomprising about 0.1 wt. % to about 50 wt. % (e.g., about 0.1 wt. % toabout 50 wt. %, about 0.5 wt. % to about 45 wt. %, about 1 wt. % toabout 40 wt. %, about 5 wt. % to about 35 wt. %, about 10 wt. % to about30 wt. %) of one or more microbially produced branched fatty alcoholsand/or derivatives thereof. An exemplary cleaning composition comprisesabout 1 wt. % to about 40 wt. % of microbially produced branched fattyalcohols and/or derivatives thereof. In another embodiment, thecomposition comprises about 2 wt. % to about 20 wt. % of microbiallyproduced branched fatty alcohols and/or derivatives thereof.

In one embodiment, the invention features a liquid cleaning compositioncomprising (a) about 0.1 wt. % to about 50 wt. % (e.g., about 0.1 wt. %to about 50 wt. %, about 0.5 wt. % to about 45 wt. %, about 1 wt. % toabout 40 wt. %, about 5 wt. % to about 35 wt. %, about 10 wt. % to about30 wt. %) of one or more microbially produced branched fatty alcoholsand/or derivatives thereof, (b) about 1 wt. % to about 30 wt. % (e.g.,about 2 wt. % to about 25 wt. %, about 5 wt. % to about 20 wt. %) of oneor more co-surfactant, (c) about 0 wt. % to about 10 wt. % (e.g., about0 wt. % to about 10 wt. %, about 0 wt. % to about 8 wt. %, about 0 wt. %to about 5 wt. %, about 0 wt. % to about 2 wt. %) of one or moredetergency builders, (d) about 0 wt. % to about 2.0 wt. % (e.g., about0.0001 wt % to about 1.5 wt. %, about 0.001 wt. % to about 1 wt. %,about 0.01 wt. % to about 0.8 wt. %) of one or more enzymes; (e) about 0wt. % to about 15 wt. % (e.g., about 0 wt. % to about 12 wt. %, about 0wt. % to about 10 wt. %, about 0 wt. % to about 8 wt. %, about 0 wt. %to about 5 wt. %) of one or more chelating agents; (f) about 0 wt. % toabout 20 wt. % (about 0 wt. % to about 15 wt. %, about 0 wt. % to about10 wt %, about 0 wt. % to about 5 wt. %) of one or more hydrotropes; (g)about 0 to about 15 wt. % (e.g., about 0 wt. % to about 10 wt. %, about0 wt. % to about 8 wt. %, about 0 wt. % to about 5 wt. %) of one or morerheology modifiers; (h) about 0 wt % to about 1.0 wt. % (e.g., about 0wt. % to about 0.8 wt. %, about 0 wt. % to about 0.5 wt. %, about 0 wt.% to about 0.2 wt. %) of one or more organic sequestering agents; and(i) about 0.1 wt. % to about 98 wt. % (e.g., about 0.1 wt. % to about 95wt. %, about 1 wt. % to about 90 wt. %, about 10 wt. % to about 85 wt.%) of a solvent system comprising water or other suitable solvents.

In another embodiment, the invention features a solid detergentcomposition comprising (a) about 0.1 wt. % to about 50 wt. % of one ormore microbially produced fatty alcohols and/or derivatives thereof, (b)about 1 wt. % to about 30 wt. % of one or more co-surfactants, (c) about1 wt. % to about 60 wt. % of one or more detergency builders, (d) about0 wt. % to about 2.0 wt. % of one or more enzymes, (e) about 0 wt. % toabout 20 wt. % of one or more hydrotropes, (f) about 10 wt. % to about35 wt. % of one or more filler salts, (f) about 0 wt. % to about 15 wt.% of one or more chelating agents, and (g) about 0.01 wt. % to about 1wt. % of one or more organic sequestering agents.

When the cleaning composition is a solid (e.g., a particulate, agranule, a tablet), the composition herein can be in any solid form,such as a granular composition or for example a tablet, flake,extrudate, agglomerate, or granule-containing composition.Alternatively, the detergent composition can be a powder. Thecomposition herein can be made by methods such as dry-mixing,agglomerating, compaction, spray drying of various ingredients comprisedin the composition herein, or a combination thereof. The compositionherein preferably has a bulk density of from about 300 g/L, 350 g/L, or450 g/L to 1500 g/L, 1000 g/L, or 850 g/L.

In certain embodiments, a liquid cleaning composition of the presentinvention is formulated such that during use, the wash water will have apH of between about 6.5 and about 11.0 (e.g., between about 6.5 to about11, between about 7.0 to about 8.5).

In one embodiment, the invention provides a dishwashing detergentcomposition. The dishwashing detergent composition can be formulated foruse in hand washing of dishes or for use in automatic dishwashers. Askilled person will appreciate that a detergent composition formulatedfor use in automatic dishwashers should contain suitable antifoamingagents in order to prevent excessive foaming of the detergentcomposition within the dishwasher. However, foaming may be desirablewhen hand washing dishes. Antifoaming agents are known. For example,various silicone antifoam compounds can be used, including a variety ofrelatively high molecular weight polymers containing siloxane units andhydrocarbyl groups of various types. Other suitable antifoam agentsinclude monocarboxylic fatty acids and soluble salts thereof, highmolecular weight fatty esters (e.g., fatty acid triglycerides), fattyacid esters of monovalent alcohols, aliphatic C₁₈-C₄₀ ketones (e.g.,stearone), N-alkylated amino triazines (e.g., tri- to hexa-alkylmelamineor di- to tetra-alkyldiamine chlortriazines), propylene oxide,bis-stearic acid amide, and monostearyl di-alkali metal (e.g., sodium,potassium, lithium) phosphate and phosphate esters, amine oxides,alkanolamides, betaines, and mixtures thereof.

In addition the dishwashing detergents can optionally comprise one ormore enzymes, gelling agents, abrasive materials, fragrances, solubilityenhancers, antideposition agents, e.g., cellulose derivatives. Abrasivematerials can be, e.g., pumice, sand, feldspar, corn meal, or mixtures.Antideposition agent can be present in an exemplary cleaning compositionin an amount of about 0.1 wt. % to about 5 wt. % (e.g., about 0.1 wt. %to about 2 wt. %).

In certain embodiments, the invention provides a laundry detergentcomposition comprising, in addition to the microbially produced branchedfatty alcohols and/or derivatives thereof as described herein, theco-surfactants and the builders, optionally one or more enzymes, gellingagents, fragrances, antideposition agents, brighteners, anticakingagents, pearlescent agents, fabric softeners, bleach systems, dyes orcolorants, preservatives, fabric care benefit agents, hueing dyes, soilrelease polymers, photoactivators, hydrolysable surfactants,anti-shrinkage agents, anti-wrinkle agents, germicides, fungicides,color speckles, colored beads, fluorinated compounds, etc.

In certain embodiments, the invention further provides a solid surfacecleaning composition. In addition to the microbially produced branchedfatty alcohols and/or derivatives thereof as described herein, theco-surfactants and the builders, the surface cleaning composition canfurther comprise one or more of the optional ingredients including,without limitation, one or more enzymes, gelling agents, fragrances,antideposition agents, pearlescent agents, soil release polymers,germicides, abrasive materials, fungicides and mixtures thereof.

In certain embodiments, the invention also provide a personal and/or petcare cleaning composition comprising one or more microbially producedbranched fatty alcohols and/or derivatives thereof, builders, andco-surfactants. Optionally, additional components can be included in thepersonal and/or pet care cleaning composition, including, for example,conditioners, silicones, silica particles, cationic cellulose or guarpolymers, silicone microemulsion stabilizers, enzymes, fattyamphiphiles, germicides, fungicides, anti-dandruff agents, pearlescentagents, foam boosters, pediculocides, pH adjusting agents, UV absorbers,sunscreens, skin active agents, vitamins, minerals, herbal/fruit/foodextracts, sphingolipids, sensory indicators, suspension agents, andmixtures thereof.

The invention further provides a method for cleaning a substrate, suchas fibers, fabrics, hard surfaces, skin, hair, etc., by contacting thesubstrate with the cleaning composition of the invention and water.Agitation is preferably provided to enhance cleaning. Suitable means forproviding agitation include rubbing by hand or with a brush, sponge,cloth, mop, or other cleaning device, automatic laundry machines,automatic dishwashers, and the like.

EXAMPLES

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only. They are not to beconstrued as limiting the scope or content of the invention in any way.

Although particular methods are described, one of ordinary skill in theart will understand that other, similar methods also can be used. Ingeneral, standard laboratory practices were used, unless otherwisestipulated. For example, standard laboratory practices were used for:cloning; manipulation and sequencing of nucleic acids; purification andanalysis of proteins; and other molecular biological and biochemicaltechniques. Such techniques are explained in detail in standardlaboratory manuals, such as Sambrook et al., Molecular Cloning: ALaboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor, New York(2000), and Ausubel et al., Current Protocols in Molecular Biology,Greene Publ. Assoc. & Wiley-Intersciences (1989).

Example 1 Constructing E. coli MG1655 ΔfadE ΔtonA AAR:Kan

This example describes the construction of a genetically engineeredmicroorganism in which the expression of a fatty acid degradation enzymeis attenuated.

The fadE gene of E. coli MG1655 was deleted using the lambda red systemdescribed by Datsenko et al., Proc. Natl. Acad. Sci. USA 97: 6640-6645(2000), with the following modifications:

The following two primers were used to create the deletion of fadE:

Del-fadE-F  (SEQ ID NO: 158)5′-AAAAACAGCAACAATGTGAGCTTTGTTGTAATTATATTGTAAACATATTGATTCCGGGGATCCGTCGACC;  and Del-fadE-R  (SEQ ID NO: 159)5′-AAACGGAGCCTTTCGGCTCCGTTATTCATTTACGCGGCTTCAACTTTCCTGTAGGCTGGAGCTGCTTC 

The Del-fadE-F and Del-fadE-R primers were used to amplify the kanamycinresistance (Km^(R)) cassette from plasmid pKD13 by PCR. The PCR productwas then used to transform electrocompetent E. coli MG1655 cellscontaining pKD46 that had been previously induced with arabinose for 3-4hours. Following a 3-hour outgrowth in SOC medium at 37° C., the cellswere plated on Luria agar plates containing 50 μg/ml of Kanamycin.Resistant colonies were identified and isolated after an overnightincubation at 37° C. Disruption of the fadE gene was confirmed in someof the colonies by PCR amplification using primers fadE-L2 and fadE-R1,which were designed to flank the E. coli fadE gene.

The fadE deletion confirmation primers were:

fadE-L2 (SEQ ID NO: 160) 5′-CGGGCAGGTGCTATGACCAGGAC;  and fadE-R1(SEQ ID NO: 161) 5′-CGCGGCGTTGACCGGCAGCCTGG 

After the fadE deletion was confirmed, a single colony was used toremove the Km^(R) marker using the pCP20 plasmid as described byDatsenko et al., supra. The resulting MG1655 E. coli strain with thefadE gene deleted and the Km^(R) marker removed was named E. coli MG1655ΔfadE, or E. coli MG 1655 D1.

Furthermore, the expression of an outer membrane protein receptor forferrichrome, colicin M, or phages T1, T5, and phi80 are attenuated.

The tonA gene of E. coli MG1655, which encodes a ferrichrome outermembrane transporter (GenBank Accession No. NP_414692), was deleted fromstrain E. coli MG1655 D1 of Example 1, using the lambda red systemaccording to Datsenko et al., supra, but with the followingmodifications:

The primers used to create the deletion of tonA were:

Del-tonA-F (SEQ ID NO: 162)5′-ATCATTCTCGTTTACGTTATCATTCACTTTACATCAGAGATATACCAATGATTCCGGGGATCCGTCGACC;  and Del-tonA-R  (SEQ ID NO: 163)5′-GCACGGAAATCCGTGCCCCAAAAGAGAAATTAGAAACGGAAGGTTGCGG TTGTAGGCTGGAGCTGCTTC 

The Del-tonA-F and Del-tonA-R primers were used to amplify the kanamycinresistance (Km^(R)) cassette from plasmid pKD13 by PCR. The PCR productobtained in this way was used to transform electrocompetent E. coliMG1655 D1 cells of Example 1 containing pKD46, which cells had beenpreviously induced with arabinose for 3-4 h. Following a 3-houroutgrowth in SOC medium at 37° C., cells were plated on Luria agarplates containing 50 μg/ml of kanamycin. Resistant colonies wereidentified and isolated after an overnight incubation at 37° C.Disruption of the tonA gene was confirmed in some of the colonies by PCRamplification using primers flanking the E. coli tonA gene: tonA-verFand tonA-verR:.

tonA-verF (SEQ ID NO: 164) 5′-CAACAGCAACCTGCTCAGCAA;  and tonA-verR(SEQ ID NO: 165) 5′-AAGCTGGAGCAGCAAAGCGTT 

After the tonA deletion was confirmed, a single colony was used toremove the Km^(R) marker using the pCP20 plasmid as described byDatsenko et al., supra. The resulting MG1655 E. coli strain having fadEand tonA gene deletions was named E. coli MG1655 ΔfadE_ΔtonA, or E. coliMG1655 DV2

The aar gene encoding Synechococcus elongatus PCC_Synpcc7942_1594 enzymeis integrated into the chromosome with the kanamycin marker directlyafter the aar sequence.

Example 2 Expression of BKD Homologs and FabH in E. coli

A branched chain alpha-keto acid dehydrogenase complex from Pseudomonasputida and a FabH from Bacillus subtilis were used to generate two E.coli plasmids for expression. First, the Pseudomonas putida BKD operonwas PCT-amplified from Pseudomonas putida F1 genomic DNA. The followingprimers were used:

P.p.BKDFUsion_F:  (SEQ ID NO: 166)5′-ATAAACCATGGATCCATGAACGAGTACGCCCC-3′ P.pBKDFusion_R:  (SEQ ID NO: 167)5′-CCAAGCTTCGAATTCTCAGATATGCAAGGCGTG-3′

Using these primers, Pseudomonas putida Pput_1450 (GenBank Accession No.A5W0E08), Pput_1451 (GenBank Accession No. A5W0E9), Pput_1452 (GenBankAccession No. A5W0F0), and Pput_1453 (A5W0F1) were amplified. The PCRproduct was then cloned into vector pGL10.173B (See, FIG. 8), a plasmidwith a pBR322 backbone and a pTrc promoter to drive gene expression. ThePCR product was cloned into pGL between BamHI and EcoRI restrictionsites. Correct insertion of the PCR product was verified by diagnosticrestriction digests. The resulting plasmid was named “pKZ4.” (See, FIG.7)

To clone E. coli PfabH promoter-B. subtilis fabH1 into a pACYC vector,insert of pDG6 (pCFDuet-E. coli PfabH promoter-B. subtilis fabH1) wassubcloned into pACYC vector using NcoI and AvrII restriction sites. Theresulting plasmid was named pDG6 (pCFDuet+E. coli PfabH+B. subtilisfabH1). (See, FIG. 6B and FIG. 6C)

E. coli strain MG1655 ΔfadE_ΔtonA, AAR:kan was transformed with pKZ4 andpDG6 (pCFDuet+E. coli PfabH+B. subtilis fabH1). The strain was evaluatedfor production of branched chain materials using shake flaskfermentation. Shake flask fermentation was carried out using Che-9media. Specifically, cultures of E. coli MG1655 ΔfadE_ΔtonA AAR:kanwithout plasmids or carrying individual plasmids were used as controls.Seed cultures of E. coli MG1655 ΔfadE_ΔtonA AAR:kan, E. coli MG1655ΔfadE_ΔtonA AAR:kan+pKZ4, E. coli MG1655 ΔfadE_ΔtonA AAR:kan+pDG6, andE. coli MG1655 ΔfadE_ΔtonA AAR:kan+pKZ4+pDG6 were grown in LB brothssupplemented with the appropriate antibiotics. After 4 hours of growth,the cultures were diluted 1:25 in Che-9 2NBT medium+appropriateselection marker and grown overnight. The cultures were then diluted in4NBT to a final OD₆₀₀˜0.2. After 6 h of growth, IPTG was added to afinal concentration of 1 mM. At 24 h post-induction, 1 mL of culture wasextracted with 0.5 mL of methyl tert-butyl ether (MTBE) and subjected toGC/MS analysis. The analysis revealed the production of iso-C_(14:0),iso-C_(15:0), anteiso-C_(15:0), iso-C_(16:0), iso-C_(17:0), andanteiso-C_(17:0) fatty alcohols. (See, FIG. 4A).

Example 3 Quantification and Identification of Branched Fatty AlcoholsInstrumentation:

The instrument is an Agilent 5975B MSD system equipped with a 30 m×0.25mm (0.10 μm film) DB-5 column. The mass spectrometer was equipped withan electron impact ionization source. Two GC/MS programs were utilized.

GC/MS program #1: The temperature of the column is held isothermal at90° C. for 5 min, then is raised to 300° C. with a 25° C./min ramp, andfinally stays at 300° C. for 1.6 min. The total run time is 15 min Withthis program, the inlet temperature is hold at 300° C. The injector isset at splitless mode. 1 μL of sample is injected for every injection.The carrier gas (helium) is released at 1.0 mL/min. The sourcetemperature of the mass spectrometer is held at 230° C.

GC/MS program #2: The temperature of the column is held isothermal at100° C. for 3 min, then is raised to 320° C. with 20° C./min, andfinally stays isothermal at 320° C. for 5 min. The total run time is 19min. The injector is set at splitless mode. 1 μL of sample is injectedfor every injection. The carrier gas (helium) is released at 1.2 mL/min.The ionization source temperature is set at 230° C.

Samples:

Extracts containing fatty alcohols by the engineered E. coli strainswere analyzed on GC/MS. In FIG. 4A chromatograms of the extracts fromthe mutant strains are compared to those from control strains which onlyproduce straight chain fatty alcohols. The branched fatty alcoholproduced are listed: iso-C_(14:0), iso-C_(15:0), anteiso-C_(15:0),iso-C_(16:0), iso-C_(17:0), and anteiso-C_(17:0).

In FIG. 4A top panel, a GC/MS chromatogram of extract from strain E.coli MG1655 ΔfadE ΔtonA AAR:kan+pKZ4+pDG6 (a) and of control strain E.coli MG1655 ΔfadE ΔtonA AAR:kan+pBR322+pCFDuet(b). Both chromatogramswere obtained with GC/MS program #2. Compared to the control strain,mutant strain produces branched-chain fatty alcohols, and the peaksrepresenting the branched fatty alcohols are boxed. GC/MSsemi-quantitative analysis:

In addition to the qualitative analysis, semi-quantitative analysis wasperformed to obtain the ratio between the branched chain compounds andthe straight chain isomers. Due to the lack of commercially availablestandards for branched fatty alcohols, accurate quantitation for thebranched chain compounds was challenging. However, by using straightchain standard with the same functional group, the relative quantity oryield of branched-chan fatty alcohols in relation to the yield of theirstraight-chain counterpart (isomers) were estimated semi-quantitatively.Standard curve quantitation method was applied, wherein standardmixtures with different concentrations were analyzed by the same GC/MSprogram as the samples. After data acquisition, the instrument response(total ion current) was plotted against the concentrations of thestandards. Linear calibration curves were obtained. (See, FIG. 5). Theconcentration of branched alcohols in a given sample was calculatedaccording to Equation 1: y=ax+b, wherein y is the instrument responsefor a particular compound in a sample. Slope a and intercept b for thiscalibration curve were determined by the linear regression of allcalibration levels of standard fatty alcohols (FIG. 4A lower panel)×(theconcentration of the branched fatty alcohol product in the sample).Accordingly, the relative concentration of branched fatty alcohols inthe production mixture was calculated.

The table below lists the compounds used as standards to quantifydifferent branched fatty alcohol compounds.

Alcohol in sample Standard used for quantitation Iso-Alc C_(15:0) AlcC_(15:0) Anteiso-Alc C_(15:0) Alc C_(15:0) Alc C_(15:0) Alc C_(15:0) AldC_(16:0) Alc C_(15:0) Alc C_(16:0) Alc C_(15:0)

Once the titers were obtained for all the fatty alcohol compounds, theratio between the production of branched chain fatty alcohols and theproduction of straight chain isomers were calculated according toequation 2:

${{Percentage}\mspace{14mu} {production}} = {\frac{{Total}\mspace{14mu} {branched}\mspace{14mu} {chain}\mspace{14mu} {products}\mspace{14mu} {in}\mspace{14mu} {mg}\text{/}L}{{Total}\mspace{14mu} {straight}\mspace{14mu} {chain}\mspace{14mu} {products}\mspace{14mu} {in}\mspace{14mu} {mg}\text{/}L} \times 100\%}$

Using this method, we were able to semi-quantitatively estimate theamount of branched fatty alcohol yield relative to the straight-chainfatty alcohol yield to be about 48%.

Example 4 Production of Branched Acyl-CoA Precursors

An E. coli strain, MG1655(DE3) ΔfadE::FRT ΔfabH::cat/pDG6 was created,which was tested for its ability to utilize branched-chain substratemolecules to create branched-chain fatty precursors of branched fattyalcohols in vivo.

The strain MG1655(DE3) ΔfadE::FRT ΔfabH::cat/pDG6 was constructed asfollows:

A region of the E. coli fabH gene described in Lai, et al., 2003, J.Biol. Chem. 278(51): 59494, was replaced by an antibiotic resistancegene. This deletion was perfomed in a strain that was complemented forfabH by the plasmid pDG6 carrying the B. subtilis fabH1 gene.

Initially, the pDG2 plasmid was constructed. The pCDFDuet-1 vector waspurchased from Novagen/EMD Biosciences. The vector carries the CloDF13replicon, lacI gene and streptomycin/spectinomycin resistance gene(aadA).

The C-terminal portion of the plsX gene, which contains an internalpromoter for the downstream fabH gene, was amplified from E. coli MG1655genomic DNA using primers 5′-TGAATTCCATGGCGCAACTCACTCTTCTTTTAGTCG-3′(SEQ ID NO:168) and 5′-CAGTACCTCGAGTCTTCGTATACATATGCGCT CAGTCAC-3′ (SEQID NO:169). These primers introduced NcoI and XhoI restriction sitesnear the ends, as well as an internal NdeI site.

Both the plsX insert and pCDFDuet-1 vector were digested withrestriction enzymes NcoI and XhoI. The cut vector was treated withAntarctic phosphatase. The insert was ligated into the vector andtransformed into chemically competent TOP10 cells. Clones were screenedby DNA sequencing. See, FIG. 6A.

Then a pDG6 plasmid was constructed using the pDG2 plasmid. The fabH1gene from Bacillus subtilis strain 168 was amplified from plasmidpLS9-114 (see, FIG. 6K) using primers 5′-CCTTGGGGCATATGAAAGCTG-3′ (SEQID NO:170) and 5′-TTTAGTCATCTCGAGTGCACCTCACCTTT-3′ (SEQ ID NO:171).These primers introduced or included NdeI and XhoI restriction sites.

Both the fabH1 insert and pDG2 vector were digested with restrictionenzymes NdeI and XhoI. The cut vector was treated with Antarcticphosphatase. The insert was ligated into the vector and transformed intochemically competent TOP10 cells. Clones were screened by DNAsequencing. See, FIG. 6B and FIG. 6C.

Then, the cat chloramphenicol resistance gene was amplified fromtemplate plasmid pKD3 using primers 5′-GCCACATTGCCGCGCCAAACGAAACCGTTTCAACCATGGCATATGAATATCCTCCTTAGTTCCTATTCCG-3′ (SEQ ID NO: 172) and5′-CGCCCCAGATTTCACGTATTGATCGGCTACGCTTAATGCAT GTGTAGGCTGGAGCTGCTTC-3′(SEQ ID NO:173) which added 50 by nucleotide ends that are homologous tothe E. coli fabH gene. This linear PCR product was used to inactivatethe E. coli fabH gene.

Strain MG1655(DE3) ΔfadE::FRT was first transformed with plasmid pKD46encoding the lambda red recombinase genes. MG1655(DE3) ΔfadE::FRT/pKD46was then transformed with plasmid pDG6. Finally, MG1655(DE3)ΔfadE::FRT/pKD46+pDG6 was induced for expression of the recombinasegenes by addition of 10 mM arabinose and transformed with the linear PCRproduct as described in Datsenko et al. (supra). Colonies were selectedon LB plates containing 30 μg/mL chloramphenicol and screened usingcolony PCR with primers 5′-TTGACACGTC TAACCCTGGC-3′ (SEQ ID NO:174) and5′-CTGTCCAGGGAACACAAATG C-3′ (SEQ ID NO:175).

A number of other constructs comprising pDG7, and pDG8 were alsoconstructed following the approach as above. The plasmids are preparedas follows.

The plasmid pDG7 was prepared from pDG2 with B. subtilis fabH2 insert.The fabH2 gene from Bacillus subtilis strain 168 was amplified fromplasmid pLS9-111 (see, FIG. 6J) using primers 5′-TTGTGTCGCCCTTTCGCTG-3′(SEQ ID NO:176) and 5′-CTTACGTACGTACTCGAGTGACGC-3′ (SEQ ID NO:177).These primers introduced or included NdeI and XhoI restriction sites.

Both the fabH2 insert and pDG2 vector were digested with restrictionenzymes NdeI and XhoI. The cut vector was treated with Antarcticphosphatase. The insert was ligated into the vector and transformed intochemically competent TOP10 cells. Clones were screened by DNAsequencing. See, FIG. 6D and FIG. 6E.

The plasmid pDG8 was prepared from pDG2 with S. coelicolor fabH insert.The fabH gene from Streptomyces coelicolor was amplified from plasmidpLS9-115 (see, FIG. 6L) using primers 5′-AAGTGGGGCATATGTCTAAGATC-3′ (SEQID NO:178) and 5′-GTGATCCGGCTCGAGGTGGTTAC-3′ (SEQ ID NO:179). Theseprimers introduced or included NdeI and XhoI restriction sites.

Both the fabH insert and pDG2 vector were digested with restrictionenzymes NdeI and XhoI. The cut vector was treated with Antarcticphosphatase. The insert was ligated into the vector and transformed intochemically competent TOP10 cells. Clones were screened by DNAsequencing. See, FIG. 6F and FIG. 6G.

The plasmid pDG10 was prepared using pCR-Blunt vector, which waspurchased from Invitrogen, with C. acetobutylicum ptb_buk operon insert,wherein the ptb part represents the gene encoding C. acetobutylicumphosphotransbutyrylase (GenBank Accession AAA75486.1, SEQ ID NO:156),and the buk part represents the gene encoding C. acetobutylicum butyratekinase (GenBank Accession JN0795, SEQ ID NO:157). The buk_ptb operon wasamplified from Clostridium acetobutylicum genomic DNA (ATCC 824) usingprimers 5′-CTTAACTTCATGTGAAAAGTTTGT-3′ (SEQ ID NO:180) and5′-ACAATACCCATGTTTATAGGGCAA-3′ (SEQ ID NO:181). The PCR product wasligated into the pCR_Blunt vector following the manufacturer'sinstructions. Colonies were verified by DNA sequencing. See, FIG. 6H andFIG. 6I.

E. coli strains were transformed with pDG10, and OP-180 plasmidcomprising E. coli thioesterase gene tesA under the control of the Ptrcpromoter and independently also one of the pDG6, pDG7 and pDG8 plasmidsas described above.

These strains were fed branched molecules isobutyrate, which resulted iniso-C_(14:0) and iso-C_(16:0) branched acyl-CoA precursors.Independently they were fed branched molecule isovalerate, whichresulted in iso-C_(13:0) and iso-C_(15:0) branched acyl-CoA precursors.See, FIG. 4B. These precursors can then be incorporated into thebranched fatty alcohol pathways as described herein and depicted in FIG.1A and FIG. 1B.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-31. (canceled) 32: A method of making a surfactant composition usingbranched chain fatty alcohols produced in a recombinant host cell, themethod comprising, (a) providing a recombinant host cell geneticallymodified to comprise (i) a polynucleotide encoding a polypeptidecomprising one or more subunits having branched chain alpha-keto aciddehydrogenase (BKD) activity (E.C. 1.2.4.4.) capable of catalyzing aconversion of an alpha-keto acid to a branched acyl-CoA, (ii) apolynucleotide encoding a polypeptide having beta-ketoacyl-ACP synthase(FabH) activity capable of catalyzing a condensation of a branchedacyl-CoA and a malonyl-ACP to produce a branched acyl-ACP, and (iii) apolynucleotide encoding a polypeptide having fatty aldehyde biosynthesisactivity capable of catalyzing a conversion of a branched fatty acyl-ACPinto a branched fatty aldehyde; (b) culturing the recombinant host cellin the presence of a carbon source under conditions effective to expressthe polynucleotides and produce branched chain fatty alcohols that aresecreted into the extracellular environment of the host cell; (c)collecting the branched chain fatty alcohols; and (d) blending thebranched chain fatty alcohols to make a surfactant composition. 33: Themethod of claim 32, further comprising a polypeptide having fattyalcohol biosynthesis activity, wherein said polypeptide is an alcoholdehydrogenase (EC 1.1.1.1).
 33. (canceled) 34: The method of claim 32,wherein said one or more subunits are selected from the group consistingof E1 alpha/beta (decarboxylase), E2 (dihydrolipoyl transacylase), andE3 (dihydrolipoyl dehydrogenase) subunits. 35: The method of claim 32,wherein said one or more subunits are selected from the group consistingof E1 alpha/beta (decarboxylase) and E2 (dihydrolipoyl transacylase)subunits. 36: The method of claim 32, wherein said polypeptide havingfatty aldehyde biosynthesis activity is an acyl-ACP reductase (AAR). 37:The method of claim 36, wherein said AAR is an enzyme from Synechococcuselongates. 38: The method of claim 32, wherein said polypeptide havingfatty aldehyde biosynthesis activity is carboxylic acid reductase (CAR).39: The method of claim 32, wherein the recombinant host cell is an E.coli host cell. 40: The method of claim 32, wherein the branched chainfatty alcohols include one or more of saturated or unsaturated C₁₂, C₁₄and C₁₆ fatty alcohols.